Colon disease targets and uses thereof

ABSTRACT

The present invention provides a method for diagnosing and detecting diseases associated with colon. The present invention provides one or more proteins or fragments thereof, peptides or nucleic acid molecules differentially expressed in colon diseases (CCAT) and antibodies binds to CCAT. The present invention provides that CCAT is used as targets for screening agents that modulates the CCAT activities. Further the present invention provides methods for treating diseases associated with colon.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. non-provisionalapplication Ser. No. 15/495,398, filed Apr. 24, 2017, which is acontinuation application of U.S. non-provisional application Ser. No.14/520,533, filed Oct. 22, 2014, which is a divisional application ofU.S. non-provisional application Ser. No. 13/621,466, filed Sep. 17,2012, which is a divisional application of U.S. non-provisionalapplication Ser. No. 12/911,617, filed Oct. 25, 2010, which is adivisional application of U.S. non-provisional application of Ser. No.12/070,716, filed Feb. 19, 2008 (issued as U.S. Pat. No. 7,842,466 onNov. 30, 2010), which is a divisional application of U.S.non-provisional application Ser. No. 11/490,374, filed Jul. 21, 2006,which claims priority to U.S. provisional application Ser. No.60/701,057, filed Jul. 21, 2005, and U.S. provisional application Ser.No. 60/717,208, filed Sep. 16, 2005, and U.S. provisional applicationSer. No. 60/763,348, filed Jan. 31, 2006, and U.S. provisionalapplication Ser. No. 60/781,313, filed Mar. 13, 2006, the contents ofeach of which are hereby incorporated by reference into thisapplication.

FIELD OF THE INVENTION

This invention relates to the fields of molecular biology and oncology.Specifically, the invention provides a molecular marker and atherapeutic agent for use in the diagnosis and treatment of colondiseases.

BACKGROUND OF THE INVENTION

Cancer currently constitutes the second most common cause of death inthe United States. Carcinomas of the colon are the eighth most prevalentform of cancer and fourth among the most common causes of cancer deathsin this country. The incidence of colon cancer has been increasingsteadily in the past twenty years in most industrialized countries,exhibiting the characteristics of a growing epidemiological problem. Inthe year 2000, for example, an estimated 28,600 deaths will be ascribedto this type of cancer and approximately 28,600 new cases will bediagnosed.

Colon cancer is the second most frequently diagnosed malignancy in theUnited States as well as the second most common cause of cancer death.The five-year survival rate for patients with colorectal cancer detectedin an early localized stage is 92%; unfortunately, only 37% ofcolorectal cancer is diagnosed at this stage. The survival rate drops to64% if the cancer is allowed to spread to adjacent organs or lymphnodes, and to 7% in patients with distant metastases.

The prognosis of colon cancer is directly related to the degree ofpenetration of the tumor through the bowel wall and the presence orabsence of nodal involvement, consequently, early detection andtreatment are especially important. Currently, diagnosis is aided by theuse of screening assays for fecal occult blood, sigmoidoscopy,colonoscopy and double contrast barium enemas. Treatment regimens aredetermined by the type and stage of the cancer, and include surgery,radiation therapy and/or chemotherapy. Recurrence following surgery (themost common form of therapy) is a major problem and is often theultimate cause of death. In spite of considerable research intotherapies for the disease, colon cancer remains difficult to diagnoseand treat. In spite of considerable research into therapies for theseand other cancers, colon cancer remains difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating such cancers. The present inventionfulfills these needs and further provides other related advantages.

In spite of considerable research into therapies for these and othercancers, colon cancer remains difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating such cancers. The present inventionfulfills these needs and further provides other related advantages suchas other colon diseases.

SUMMARY OF THE INVENTION

The present invention is based on the identification of certain cellsurface proteins, cytosolic proteins, and/or secreted proteins that aredifferentially expressed in colon cancer. A malignant cell often differsfrom a normal cell by a differential expression of one or more proteins.These differentially expressed proteins, and the fragments thereof, areimportant markers for the diagnosis of colon disease. The differentiallyexpressed proteins of the present invention and the nucleic acidsencoding said proteins and the fragments of said proteins are referredto herein as colon cancer associated target, CCAT proteins or CCATnucleic acids or CCAT peptides, respectively.

The present invention provides peptides and protein differentiallyexpressed in colon diseases (hereinafter CCAT). Based on the site ofprotein localization, e.g., surface or cytosolic, and proteincharacterization, e.g. receptor or enzyme, specific uses of these CCATsare provided. Some of the CCATs of the present invention serve astargets for one or more classes of theraputic agents, while others maybe suitable for antibody theraputics.

Accordingly, the present invention provides a method for diagnosing ordetecting colon disease in a subject comprising: determining the levelof one or more CCAT proteins, or any fragment(s) thereof, in a testsample from said subject, wherein said CCAT protein comprises a sequenceselected from a group consisting of SEQ ID NOS:1-1233; wherein adifferential level of said CCAT protein(s) or fragment(s) in said samplerelative to the level of said protein(s) or fragment(s) in a test samplefrom a healthy subject, or the level established for a healthy subject,is indicative of colon disease.

The present invention also provides a method for detecting colon cancerin a subject comprising: determining the level of one or more CCATpeptide(s) comprising a peptide sequence selected from a groupconsisting of SEQ ID NOS:2228-2638 in a test sample from said subject,wherein a differential level of said CCAT peptide(s) in said sample tothe level of said CCAT peptide(s) in a test sample from a healthysubject, or the level of said CCAT peptide(s) established for a healthysubject, is indicative of colon disease.

The present invention further provides a method for detecting colondisease in a subject comprising: determining the level of one or moreCCAT nucleic acid(s), or any fragment(s) thereof, in a test sample fromsaid subject, wherein said CCAT nucleic acid(s) encode a CCAT proteinsequence selected from a group consisting of SEQ ID NOS:1-1233; whereina differential level of said CCAT nucleic acids or fragment(s) in saidsample relative to the level of said protein(s) or fragment(s) in a testsample from a healthy subject, or the level established for a healthysubject, is indicative of colon disease.

The invention also provides methods for detecting the CCAT peptides,gene or mRNA in a test sample for use in diagnosing the presence,absence or progression of a disease. The test sample includes but is notlimited to a biological sample such as tissue, blood, serum orbiological fluid.

The present invention further provides a purified antibody that bindsspecifically to a protein molecule, or any fragment thereof, selectedfrom a group consisting of SEQ ID NOS:1-1233.

The present invention further provides a composition comprising anantibody that binds to a protein selected from a group consisting of SEQID NOS:1-1233 and an acceptable carrier.

The present invention further provides a method for treating colondisease, comprising administering to a patient in need of said treatmenta therapeutically effective amount of one or more antibody(ies) of thisinvention.

The present invention further provides a method for treating colondisease comprising (i) identifying a subject having colon disease and(ii) administering to a said patient a therapeutically effective amountof one or more antibody(ies) of this invention.

The present invention further provides a method to screen for agentsthat modulate CCAT protein activity, comprising the steps of (i)contacting a test agent with a CCAT protein and (ii) assaying for CCATprotein activity, wherein a change in said activity in the presence ofsaid agent relative to CCAT protein activity in the absence of saidagent indicates said agent modulates said CCAT protein activity.

The present invention further provides a method to screen for agentsthat bind to CCAT protein, comprising the steps of (i) contacting a testagent with a CCAT protein and (ii) measuring the level of binding ofagent to said CCAT protein.

The invention also provides diagnostic methods for human disease, inparticular for colon diseases, its metastatic stage, and therapeuticpotential.

The present invention further provides diagnostic method forepithelial-cell related cancers. In particular pancreas, lung, colon,prostate, ovarian, breast, bladder renal, hepatocellular, pharyngeal,and gastric cancer.

The invention also provides a method for monitoring the diseaseprogression and the treatment progress.

The invention further provide a method of diagnosis by an array, whereinthe array is immobilized with two or more CCAT proteins, peptides ornucleic acid molecules. The proteins, peptides or nucleic acid moleculesinclude but are not limited to the SEQ ID NOS: 1-2638.

The invention also provides monoclonal or polyclonal antibodies andcomposition thereof reactive with antigentic portion of CCAT protein,peptides or fragments thereof in a form for use in colon diseasesdiagnosis.

The invention further provides an immunogenic antibody for treatingcolon diseases disease or diseases associated with colon diseases.

The present invention provides a method for screening agents thatmodulate CCAT activity, comprising the steps of (a) contacting a samplecomprising CCAT with an agent; and (b) assaying for CCAT activity,wherein a change in said CCAT activity in the presence of said agentrelative to CCAT activity in the absence of said compound indicates saidagent modulates CCAT. The agents include but are not limited to protein,peptide, antibody, nucleic acid such as antisense RNA, RNAi fragments,small molecules.

The present invention further provides a method for treating colondiseases, comprising: administering to a patient one or more agents in atherapeutically effective amount to treat colon diseases.

The present invention provides a method for treating colon diseases,comprising: identifying a subject having colon diseases; andadministering to a patient to one or more antibodies in atherapeutically effective amount to treat colon diseases.

The present invention further provides therapeutic potential forepithelial-cell related cancers. In particular pancreas, lung, colon,prostate, ovarian, breast, bladder renal, hepatocellular, pharyngeal andgastric cancer.

Description of the Files Contained on the CD-R Named CL001615CDR

The CD-R named CL001615CDR contains the following three text (ASCII)files:

1) File SEQLIST_1615.txt provides the Sequence Listing. The SequenceListing provides the protein sequences (SEQ ID NOS:1-1233); transcriptsequences (SEQ ID NOS:1234-2227) and peptide sequences (SEQ IDNOS:2228-2638) as shown in Table 1. File SEQLIST_1615.txt is 11,250 KBin size.2) File TABLE1_1615.txt provides Table 1. File TABLE1_1615 is 402 KB insize.3) File TABLE2_1615.txt provides Table 2. File TABLE2_1615 is 5 KB insize.

The material contained on the CD-R labeled CL001615CDR is herebyincorporated by reference pursuant to 37 CFR 1.77(b)(4).

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210017259A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Description of Table 1

Table 1 (provided on the CD-R) discloses the peptides which correspondto the protein in the colon cancer tumor, the expression information,and the ratio compare to the control sample. The expression is based onmeasuring the level of the peptides. “N/A” represents the number ofoverexpression by more than two, whereas numerical representation ofoverexpression is also indicated. “S” which is overexpressed singletonindicates that the peptide peak in diseased sample was detected andthere was no peak detected in control samples.

Table 1 also discloses the CCAT proteins, transcripts, and peptides.

The transcript/protein information includes:

-   -   a protein number (1 through 1233)    -   a Celera protein internal identification number for the protein        encoded by the Celera transcript (hCP and/or UID)    -   a public protein accession number (Genbank e.g., RefSeq NP        number, Swiss-prot, or Derwent) for the protein    -   an art-known gene/protein name    -   a Celera transcript internal identification number (hCT and/or        UID)    -   a public transcript accession number (Genbank e.g., RefSeq NM        number, or Derwent)    -   a Celera hCG and UID internal identification numbers for the        gene    -   an art-known gene symbol    -   Celera genomic axis position (indicating start nucleotide        position-stop nucleotide position)    -   the chromosome number of the chromosome on which the gene is        located    -   an OMIM (Online Mendelian Inheritance in Man; Johns Hopkins        University/NCBI) public reference number for obtaining further        information regarding the medical significance of each gene    -   alternative gene/protein name(s) and/or symbol(s) in the OMIM        entry

Description of Table 2

Table 2 discloses tumor staging information as follows (all tumorstaging designations in Table 2 are those which are typically used inthe art): Sample ID Number, sample type (labeled “Sample”), cancer type(labeled “Type”), Lymph Nodes (the “N” in the TNM staging system, forNode, which designates the spread of a tumor to the lymph nodes),Distant Metastasis (the “M” in the TNM staging system, for Metastasis,which designates the extent of tumor metastasis), Extent of Invasion(the “T” in the TNM staging system, for Tumor, which designates the sizeand location of a primary tumor), and AJCC Stage (which is assignedbased on a combination of the T, N, and M classifications).

The Lymph Nodes (“N”) stages are as follows: “NX”=the regional lymphnodes cannot be assessed, “NO”=the cancer has not spread into theregional lymph nodes, “N1”=the cancer has spread to one to threeregional lymph nodes, and “N2”=the cancer has spread to four or moreregional lymph nodes.

The Distant Metastasis (“M”) stages are as follows: “MX”=distant spreadcannot be assessed, “M0”=the disease has not metastasized, and“M1”=there is metastasis to another part of the body beyond the colon orrectum.

The Extent of Invasion (“T”) stages are as follows: “TX”=the primarytumor cannot be evaluated, “T0”=there is no evidence of a primary tumorin the colon or rectum, “Tis”=carcinoma in situ (cancer cells arepresent only in the epithelium or lamina propria), “T1”=the tumorextends into the submucosa of the colon, “T2”=the tumor has invaded themuscularis propria, “T3”=the tumor has grown through the muscularispropria and into the subserosa or into tissues surrounding the colon orrectum, and “T4”=the tumor has invaded other organs beyond the colon orrectum or has caused a perforation in the wall of the colon or rectum.

The AJCC Stages are as follows: “0”=carcinoma in situ, “I”=the cancerhas grown through the mucosa and has invaded the muscular layer of thecolon or rectum but has not spread into nearby tissues or lymph nodes(T1 or T2, N0, M0), “IIA”=the cancer has spread through the wall of thecolon or rectum and may have spread to nearby tissues but has not spreadto the nearby lymph nodes (T3, N0, M0), “IIB”=the cancer has spreadthrough the colon or rectum to nearby organs but has not spread to thenearby lymph nodes (T4, N0, M0), “IIIA”=the cancer has grown through theinner lining or into the muscle layers of the intestine and to one tothree lymph nodes, but has not spread to other parts of the body (T1 orT2; N1, M0), “IIIB”=the cancer has grown through the bowel wall or tosurrounding organs and into one to three lymph nodes, but has not spreadto other parts of the body (T3 or T4, N1, M0), “IIIC”=the cancer (whichcan be of any size) has spread to four or more lymph nodes, but not toother distant parts of the body (any T, N2, M0), “IV”=the cancer hasmetastasized to distant parts of the body (e.g., liver or lungs) (any T,any N, M1), and “Recurrent”=the cancer has returned to the colon orrectum or another part of the body following treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS General Description

While the broadest definition of this invention is set forth in theSummary of the Invention, certain nucleic acids, peptides or proteinsare preferred. For example a preferred method for detecting colondisease by determining the level of one or more CCAT protein(s) or anyfragment(s) thereof is wherein the level of CCAT protein(s) aredetermined by contacting one or more antibody(ies) that specificallybind to the antigenic regions of the CCAT protein(s). Further preferredis a method wherein the level of two or more proteins are determined,more preferred wherein the level of four or more proteins are determinedand most preferred wherein the level of eight or more proteins aredetermined.

A preferred method for detecting colon disease by determining the levelof one or more CCAT peptide(s) is wherein the level of CCAT peptides(s)are determined by contacting one or more antibody(ies) that specificallybind to the antigenic regions of the CCAT peptide(s). Further preferredis a method wherein the level of five or more peptides are determined,more preferred wherein the level of ten or more peptides are determinedand most preferred wherein the level of fifteen or more peptides aredetermined.

A preferred method for detecting colon disease by determining the levelof one or more CCAT nucleic acid(s) is wherein the level of said CCATnucleic acid(s) is determined by contacting one or more probes thatspecifically hybridize to said nucleic acid(s). Further preferred is amethod wherein the level of two or more nucleic acids are determined,more preferred wherein the level of four or more nucleic acids aredetermined and most preferred wherein the level of eight or more nucleicacids are determined.

The methods for detecting colon disease provided by the presentinvention may be used for diagnosing the presence of disease in apatent, monitoring the presence of colon disease in patients undergoingtreatment and testing for the reoccurrence of colon disease in patientsthat were successfully treated for colon disease; preferably wherein thecolon disease is colon cancer. The test sample may be, but is notlimited to, a biological sample such as tissue, blood, serum orbiological fluid.

The present invention is based on the discovery of protein(s) andpeptide(s) that are differentially expressed in colon cancer samplesversus normal colon diseases samples. These proteins and peptide, andthe encoding nucleic acid molecules are associated with colon diseases,hereinafter the CCAT protein, peptide or nucleic acids.

The discovery of disease specific target proteins is base on discoveriesmade using proteomics techniques. The method uses on MALDI-TOF TOF LC/MSanalyses platform to generate protein expression profiles from colondiseases tissues or cell lines in an effort to discover and identifynovel molecules associated with the disease.

Based on these discoveries, the present invention provides proteins,peptides, nucleic acids that are differential in colon diseases, as wellas antibodies binds to the proteins or peptides. The present inventionalso provides methods for detection, monitoring, diagnosis, prognosis,preventive and treatment of colon diseases. The present inventionprovides a detection reagent, markers for colon diseases at variousstages, comprises CCAT sequences isolated from human colon diseasestissue, sera, cell lines, blood or biological fluids.

The present invention provides a method for treating colon diseasestargeting at CCAT. The treatment includes administration of atherapeutically effective amount of composition comprise, but not limitto, an antibody, an immunogentic peptide which induces T cell response,a small molecule, a protein or a nucleic acid molecule. The compositionfurther comprises an agonist or antagonist to CCAT. A “Colon orcolorectal disease” includes but not limited to colon cancer, colontumor, diverticulosis, diverticulitis, Crohn's disease, ulcerativecolitis, irritable bowel syndrome, inflammatory bowel disease,hemorrhoids, and anal fissure.

The present invention may further provide a diagnostic or therapeuticpotential for epithelial-cell related cancers, which include but are notlimited to pancreas, lung, colon, prostate, ovarian, breast, bladderrenal, hepatocellular, pharyngeal and gastric cancers.

The present invention further provides the target for screening an agentfor CCAT, wherein the agent is compounds of small molecules, proteins,peptides, nucleic acids, antibodies or other agonists or antagonists.

CCAT Peptide/Proteins and Peptides

The present invention provides isolated CCAT peptide and proteinmolecules that consisting of, consisting essentially of, or comprisingthe amino acid sequences of the CCAT peptides and proteins disclosed inTable 1, (encoded by the nucleic acid molecule shown in Table 1), aswell as all obvious variants of these peptides that are within the artto make and use. Some of these variants are described in detail below.

In one embodiment CCAT peptides include, but are not limited to, theamino acid sequence of SEQ ID NOS:2228-2638 and variants thereof. A CCATprotein includes, but is not limited to, the amino acid sequence of SEQID NOS:1-1233 and variants thereof. CCAT proteins may be differentiallyexpressed in colon cell line, blood, tissue, serum or body fluids.

The peptide or protein or fragment thereof, to which the inventionpertains, however, are not to be construed as encompassing peptide,protein or fragment that may be disclosed publicly prior to the presentinvention.

The CCAT proteins and peptides of the present invention can be purifiedto homogeneity or other degrees of purity. The level of purificationwill be based on the intended use. The critical feature is that thepreparation allows for the desired function of the peptide, even if inthe presence of considerable amounts of other components (the featuresof an isolated nucleic acid molecule is discussed below).

As used herein, a “peptide” is defined as amino acid sequences between5-20 amino acids derived from CCAT proteins such as SEQ ID NOS:1-1233 orvariants thereof. The peptide differentially expressed in either colondiseases cell line, blood, tissue, serum or body fluids. In oneembodiment peptides include, but are not limited to, the amino acidsequence of SEQ ID NOS:2228-2638, or variants thereof.

As used herein, a “protein” is full-length protein differentiallyexpressed in colon diseases cell line, tissue, blood, serum or bodyfluids. A protein includes, but is not limited to, the amino acidsequence of SEQ ID NOS:1-1233.

A peptide is said to be “isolated” or “purified” when it issubstantially free of cellular material or free of chemical precursorsor other chemicals. The peptides of the present invention can bepurified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule are discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theCCAT peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated CCAT proteins and peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd.ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001). Experimental data as provided in Table 1 indicates expression inhuman colon tumor tissues. For example, a nucleic acid molecule encodingthe CCAT protein or peptide is cloned into an expression vector, theexpression vector introduced into a host cell and the protein expressedin the host cell. The protein or peptide can then be isolated from thecells by an appropriate purification scheme using standard proteinpurification techniques. Many of these techniques are described indetail below.

A CCAT peptide or protein can be attached to heterologous sequences toform chimeric or fusion proteins. Such Schimeric and fusion proteinscomprise a peptide operatively linked to a heterologous protein havingan amino acid sequence not substantially homologous to the peptide.“Operatively linked” indicates that the peptide and the heterologousprotein are fused in-frame. The heterologous protein can be fused to theN-terminus or C-terminus of the peptide.

In some uses, the fusion protein does not affect the activity of thepeptide or protein per se. For example, the fusion protein can include,but is not limited to, fusion proteins, for example beta-galactosidasefusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged,HI-tagged and Ig fusions. Such fusion proteins, particularly poly-Hisfusions, can facilitate the purification of recombinant CCAT proteins orpeptides. In certain host cells (e.g., mammalian host cells), expressionand/or secretion of a protein can be increased by using a heterologoussignal sequence.

A chimeric or fusion CCAT protein or peptide can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A CCAT-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the CCAT protein or peptide.

As mentioned above, the CCAT peptide or the CCAT protein has obviousvariants of the amino acid sequence, such as naturally occurring matureforms of the CCAT, allelic/sequence variants of the CCAT, non-naturallyoccurring recombinantly derived variants of the CCATs, and orthologs andparalogs of the CCAT proteins or peptides. Such variants can readily begenerated using art-known techniques in the fields of recombinantnucleic acid technology and protein biochemistry.

It is understood, however, that CCAT and variants exclude any amino acidsequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the CCAT peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package, usingeither a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Inyet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1):387(1984)), using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In anotherembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NB LAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of theCCAT peptides of the present invention as well as being encoded by thesame genetic locus as the CCAT peptide provided herein (see Table 1).

Allelic variants of a CCAT peptide can readily be identified as being ahuman protein having a high degree (significant) of sequencehomology/identity to at least a portion of the CCAT peptide as well asbeing encoded by the same genetic locus as the CCAT peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in Table 1, such as the genomic sequence mapped tothe reference human. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a CCAT peptide encodingnucleic acid molecule under stringent conditions as more fully describedbelow.

Paralogs of a CCAT peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the CCAT peptide, as being encoded by a gene from humans, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a CCAT peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a CCAT peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the CCAT peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from mammals, preferablyprimates, for the development of human therapeutic targets and agents.Such orthologs will be encoded by a nucleic acid sequence that willhybridize to a CCAT peptide encoding nucleic acid molecule undermoderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the CCAT peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the CCAT peptide. Forexample, one class of substitutions is conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a CCAT peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

Variant CCAT peptides can be fully functional or can lack function inone or more activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as CCAT activity or in assays such as an invitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

The present invention further provides fragments of the CCATs, inaddition to proteins and peptides that comprise and consist of suchfragments, particularly those comprising the residues identified inTable 1. As used herein, a fragment comprises at least 8, 10, 12, 14,16, 18, 20 or more contiguous amino acid residues from a CCAT. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the CCAT or could be chosen for the abilityto perform a function, e.g. bind a substrate or act as an immunogen.Particularly important fragments are biologically active fragments,peptides that are, for example, about 8 or more amino acids in length.Such fragments will typically comprise a domain or motif of the CCAT,e.g., active site, a transmembrane domain or a substrate-binding domain.Further, possible fragments include, but are not limited to, domain ormotif containing fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in CCATs are described inbasic texts, detailed monographs, and the research literature, and theyare well known to those of skill in the art.

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the CCATs of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which the mature CCAT is fused with another compound, suchas a compound to increase the half-life of the CCAT (for example,polyethylene glycol), or in which the additional amino acids are fusedto the mature CCAT, such as a leader or secretory sequence or a sequencefor purification of the mature CCAT or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in Table1; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a CCAT-effectorprotein interaction or CCAT-ligand interaction), the protein can be usedto identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, Sambrook, J., E. F. Fritschand T. Maniatis eds., 3rd. ed., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (2001) and “Methods in Enzymology: Guide toMolecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R.Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, CCATs isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the CCAT. Experimental data as provided inTable 1 indicate that the CCAT of the present invention areoverexpressed in colon tumor tissue. A large percentage ofpharmaceutical agents are being developed that modulate the activity ofCCAT proteins, particularly members of the CCAT subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in Table 1.Experimental data as provided in Table 1 indicates expression in humancolon tumor tissues. Such uses can readily be determined using theinformation provided herein, that which is known in the art, and routineexperimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to CCATs that are related to members ofthe CCAT subfamily. Such assays involve any of the known CCAT functionsor activities or properties useful for diagnosis and treatment ofCCAT-related conditions that are specific for the subfamily of CCATsthat the one of the present invention belongs to, particularly in cellsand tissues that express the CCAT. Experimental data as provided inTable 1 indicate that the CCAT of the present invention areoverexpressed in colon tumor tissue

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the CCAT, as a biopsy orexpanded in cell culture. Experimental data as provided in Table 1indicates expression in human colon tumor tissues. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe CCAT protein.

The polypeptides can be used to identify compounds or agents thatmodulate CCAT activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with theCCAT. Both the CCATs of the present invention and appropriate variantsand fragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the CCAT. These compounds can befurther screened against a functional CCAT to determine the effect ofthe compound on the CCAT activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the CCAT to a desired degree.

Further, the proteins of the present invention can be used to screen acompound or an agent for the ability to stimulate or inhibit interactionbetween the CCAT protein and a molecule that normally interacts with theCCAT protein, e.g. a substrate or or an extracellular binding ligand ora component of the signal pathway that the CCAT protein normallyinteracts (for example, a cytosolic signal protein or another CCAT).Such assays typically include the steps of combining the CCAT proteinwith a candidate compound under conditions that allow the CCAT protein,or fragment, to interact with the target molecule, and to detect theformation of a complex between the protein and the target or to detectthe biochemical consequence of the interaction with the CCAT protein andthe target, such as any of the associated effects of signal transductionsuch as protein phosphorylation, cAMP turnover, and adenylate cyclaseactivation, etc.

Candidate compounds or agents include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)2, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound or agent is a soluble fragment of the CCAT thatcompetes for substrate binding. Other candidate compounds include mutantCCATs or appropriate fragments containing mutations that affect CCATfunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) CCAT activity. The assaystypically involve an assay of events in the signal transduction pathwaythat indicate CCAT activity. Thus, the phosphorylation of a substrate,activation of a protein, a change in the expression of genes that areup- or down-regulated in response to the CCAT protein dependent signalcascade can be assayed.

Any of the biological or biochemical functions mediated by the CCAT canbe used as an endpoint assay. These include all of the biochemical orbiochemical/biological events described herein, in the references citedherein, incorporated by reference for these endpoint assay targets, andother functions known to those of ordinary skill in the art or that canbe readily identified using the information provided in Table 1.Specifically, a biological function of a cell or tissues that expressesthe CCAT can be assayed. Experimental data as provided in Table 1indicate that the CCAT of the present invention are overexpressed incolon tumor tissue.

Binding and/or activating compounds can also be screened by usingchimeric CCAT proteins in which the amino terminal extracellular domain,or parts thereof, the entire transmembrane domain or subregions, such asany of the seven transmembrane segments or any of the intracellular orextracellular loops and the carboxy terminal intracellular domain, orparts thereof, can be replaced by heterologous domains or subregions.For example, a substrate-binding region can be used that interacts witha different substrate then that which is recognized by the native CCAT.Accordingly, a different set of signal transduction components isavailable as an end-point assay for activation. This allows for assaysto be performed in other than the specific host cell from which the CCATis derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the CCAT (e.g. binding partners and/or ligands). Thus, a compoundis exposed to a CCAT polypeptide under conditions that allow thecompound to bind or to otherwise interact with the polypeptide. SolubleCCAT polypeptide is also added to the mixture. If the test compoundinteracts with the soluble CCAT polypeptide, it decreases the amount ofcomplex formed or activity from the CCAT. This type of assay isparticularly useful in cases in which compounds are sought that interactwith specific regions of the CCAT. Thus, the soluble polypeptide thatcompetes with the target CCAT region is designed to contain peptidesequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the CCAT protein, or fragment, or its target moleculeto facilitate separation of complexes from uncomplexed forms of one orboth of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofCCAT-binding protein found in the bead fraction quantitated from the gelusing standard electrophoretic techniques. For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a CCAT-binding proteinand a candidate compound are incubated in the CCAT protein-presentingwells and the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the CCAT protein targetmolecule, or which are reactive with CCAT protein and compete with thetarget molecule, as well as CCAT-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

Agents that modulate one of the CCATs of the present invention can beidentified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of CCAT protein activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedby the CCAT pathway, by treating cells or tissues that express the CCAT.Experimental data as provided in Table 1 indicates expression in humancolon tumor tissues. These methods of treatment include the steps ofadministering a modulator of CCAT activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

In yet another aspect of the invention, the CCAT proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with the CCAT and are involved in CCAT activity. SuchCCAT-binding proteins are also likely to be involved in the propagationof signals by the CCAT proteins or CCAT targets as, for example,downstream elements of a CCAT-mediated signaling pathway. Alternatively,such CCAT-binding proteins are likely to be CCAT inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a CCAT protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences that encode an unidentified protein (“prey” or“sample”) is fused to a gene that codes for the activation domain of theknown transcription factor. If the “bait” and the “prey” proteins areable to interact, in vivo, forming a CCAT-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the CCATprotein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a CCAT-modulating agent, an antisense CCATnucleic acid molecule, an CCAT-RNAi fragment, a CCAT-specific antibody,or a CCAT-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

The CCAT proteins of the present invention are also useful to provide atarget for diagnosing a disease or predisposition to disease mediated bythe peptide. Accordingly, the invention provides methods for detectingthe presence, or levels of, the protein (or encoding mRNA) in a cell,tissue, or organism. Experimental data as provided in Table 1 indicatesexpression in human colon tumor tissues. The method involves contactinga biological sample with a compound capable of interacting with the CCATprotein such that the interaction can be detected. Such an assay can beprovided in a single detection format or a multi-detection format suchas an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered CCAT activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes affects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the CCAT protein in which one ormore of the CCAT functions in one population are different from those inanother population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and CCAT activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in Table 1 indicates expression in humancolon tumor tissues. Accordingly, methods for treatment include the useof the CCAT protein or fragments.

Antibodies

The present invention provides antibodies specifically bind to CCATproteins or fragments thereof, peptides, or antigenic portion thereof.

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof as describe above.

The antibody of present invention selectively binds a target CCAT whenit binds the target domain and does not significantly bind to unrelatedproteins. An antibody is still considered to selectively bind a peptideeven if it also binds to other proteins that are not substantiallyhomologous with the target peptide so long as such proteins sharehomology with a fragment or domain of the peptide target of theantibody. In this case, it would be understood that antibody binding tothe peptide is still selective despite some degree of cross-reactivity.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), humanized antibody and antibody fragments (e.g.,Fab, F(ab′).sub.2 and Fv) so long as they exhibit the desired biologicalactivity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity.

As used herein, antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (VH) followed by a number of constant domains. Each light chainhas a variable domain at one end (VL) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light andheavy chain variable domains. Chothia et al., J. Mol. Biol. 186, 651-63(1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596(1985).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of the environment in which isproduced. Contaminant components of its production environment arematerials that would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In preferred embodiments, the antibody willbe purified as measurable by at least three different methods: 1) togreater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight; 2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequentator; or 3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomasie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “antigenic region” or “antigenic determinant” or an “epitope”includes any protein determinant capable of specific binding to anantibody. This is the site on an antigen to which each distinct antibodymolecule binds. Epitopic determinants usually consist of active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three-dimensional structural characteristics, aswell as charge characteristics.

“Antibody specificity,” is an antibody, which has a stronger bindingaffinity for an antigen from a first subject species than it has for ahomologue of that antigen from a second subject species. Normally, theantibody “bind specifically” to a human antigen (i.e., has a bindingaffinity (Kd) value of no more than about 1×10⁻⁷ M, preferably no morethan about 1×10⁻⁸ M and most preferably no more than about 1×10⁻⁹ M) buthas a binding affinity for a homologue of the antigen from a secondsubject species which is at least about 50 fold, or at least about 500fold, or at least about 1000 fold, weaker than its binding affinity forthe human antigen. The antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody (Queen et al., U.S. Pat. Nos. 5,530,101, 5, 585,089; 5,693,762;and 6,180,370).

The present invention provides an “antibody variant,” which refers to anamino acid sequence variant of an antibody wherein one or more of theamino acid residues have been modified. Such variant necessarily haveless than 100% sequence identity or similarity with the amino acidsequence having at least 75% amino acid sequence identity or similaritywith the amino acid sequence of either the heavy or light chain variabledomain of the antibody, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, and most preferably at least95%. Since the method of the invention applies equally to bothpolypeptides, antibodies and fragments thereof, these terms aresometimes employed interchangeably.

The term “variable” in the context of variable domain of antibodiesrefers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs) also known as hypervariableregions both in the light chain and the heavy chain variable domains.There are at least two techniques for determining CDRs: (1) an approachbased on cross-species sequence variability (i.e., Kabat et al.,Sequences of Proteins of Immunological Interest (National Institute ofHealth, Bethesda, Md. 1987); and (2) an approach based oncrystallographic studies of antigen-antibody complexes (Chothia, C. etal. (1989), Nature 342: 877). The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a .beta.-Sheet configuration, connected by three CDRs, whichform loops connecting, and in some cases forming part of, the.beta.-sheet structure. The CDRs in each chain are held together inclose proximity by the FR regions and, with the CDRs from the otherchain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al.) The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments which are capable of crosslinkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen recognition and binding site. This region consists of adimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site.

The Fab fragment [also designated as F(ab)] also contains the constantdomain of the light chain and the first constant domain (CH1) of theheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains have a free thiol group. F(ab′) fragments areproduced by cleavage of the disulfide bond at the hinge cysteines of theF(ab′)₂ pepsin digestion product. Additional chemical couplings ofantibody fragments are known to those of ordinary skill in the art.

The present invention further provides monoclonal antibody, polyclonalantibody as well as humanized antibody. In general, to generateantibodies, an isolated peptide is used as an immunogen and isadministered to a mammalian organism, such as a rat, rabbit or mouse.The full-length protein, an antigenic peptide fragment or a fusionprotein of the CCAT protein can be used. Particularly importantfragments are those covering functional domains, some but not all theexamples of the domains are identified in Table 1. Many methods areknown for generating and/or identifying antibodies to a given targetpeptide. Several such methods are described by Harlow, Antibodies, ColdSpring Harbor Press, (1989).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”antibody indicates the character of the antibody as being obtained froma substantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature 256, 495 (1975), or may be madeby recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.The monoclonal antibodies for use with the present invention may also beisolated from phage antibody libraries using the techniques described inClackson et al. Nature 352: 624-628 (1991), as well as in Marks et al.,J. Mol. Biol. 222: 581-597 (1991). For detailed procedure for making amonoclonal antibody, see the Example below.

“Humanized” forms of non-human (e.g. murine or rabbit) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibody may comprise residues, which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., Nature 321,522-525 (1986); Reichmann et al., Nature 332, 323-327 (1988) and Presta,Curr. Op. Struct. Biol. 2, 593-596 (1992).

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen such as CCAT protein,peptides or fragments thereof and a carrier protein is prepared and ananimal is immunized by the complex according to the same manner as thatdescribed with respect to the above monoclonal antibody preparation andthe description in the Example. A serum or plasma containing theantibody against the protein is recovered from the immunized animal andthe antibody is separated and purified. The gamma globulin fraction orthe IgG antibodies can be obtained, for example, by use of saturatedammonium sulfate or DEAE Sephadex, or other techniques known to thoseskilled in the art.

The antibody titer in the antiserum can be measured according to thesame manner as that described above with respect to the supernatant ofthe hybridoma culture. Separation and purification of the antibody canbe carried out according to the same separation and purification methodof antibody as that described with respect to the above monoclonalantibody and in the Example.

The protein used here in as the immunogen is not limited to anyparticular type of immunogen. In one aspect, antibodies are preferablyprepared from regions or discrete fragments of the CCAT proteins.Antibodies can be prepared from any region of the peptide as describedherein. In particular, they are selected from a group consisting of SEQID NOS:2228-2638 and fragments of SEQ ID NOS:1-1233. An antigenicfragment will typically comprise at least 8 contiguous amino acidresidues. The antigenic peptide can comprise, however, at least 10, 12,14, 16 or more amino acid residues. Such fragments can be selected on aphysical property, such as fragments correspond to regions that arelocated on the surface of the protein, e.g., hydrophilic regions or canbe selected based on sequence uniqueness.

Antibodies may also be produced by inducing production in the lymphocytepopulation or by screening antibody libraries or panels of highlyspecific binding reagents as disclosed in Orlandi et al. (1989; ProcNatl Acad Sci 86:3833-3837) or Winter et al. (1991; Nature 349:293-299).A protein may be used in screening assays of phagemid or B-lymphocyteimmunoglobulin libraries to identify antibodies having a desiredspecificity. Numerous protocols for competitive binding or immunoassaysusing either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Smith G. P., 1991, Curr. Opin.Biotechnol. 2: 668-673.

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

Antibody can be also made recombinantly. When using recombinanttechniques, the antibody variant can be produced intracellularly, in theperiplasmic space, or directly secreted into the medium. If the antibodyvariant is produced intracellularly, as a first step, the particulatedebris, either host cells or lysed fragments, is removed, for example,by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which aresecreted to the periplasmic space of E. coli. Briefly, cell paste isthawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody variant is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibodies or antigen binding fragments may also be produced bygenetic engineering. The technology for expression of both heavy andlight cain genes in E. coli is the subject the following PCT patentapplications; publication number WO 901443, WO901443, and WO 9014424 andin Huse et al., 1989 Science 246:1275-1281. The general recombinantmethods are well known in the art.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel elecrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human .delta.1, .delta.2or .delta.4 heavy chains (Lindmark et al., J. Immunol Meth. 62: 1-13(1983)). Protein G is recommended for all mouse isotypes and for human.delta.3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH3 domain, the Bakerbond ABXTM resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in Table 1 indicate that theCCAT of the present invention are overexpressed in colon tumor tissue.Further, such antibodies can be used to detect protein in situ, invitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression. Also, such antibodies can be usedto assess abnormal tissue distribution or abnormal expression duringdevelopment or progression of a biological condition. Antibody detectionof circulating fragments of the full length protein can be used toidentify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in Table 1 indicatesexpression in human colon tumor tissues. If a disorder is characterizedby a specific mutation in the protein, antibodies specific for thismutant protein can be used to assay for the presence of the specificmutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in Table 1 indicates expression in humancolon tumor tissues. The diagnostic uses can be applied, not only ingenetic testing, but also in monitoring a treatment modality.Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy. More detection and diagnosis methods are describedin detail below.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in Table 1 indicates expression in human colon tumor tissues.Thus, where a specific protein has been correlated with expression in aspecific tissue, antibodies that are specific for this protein can beused to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the CCAT peptide to a binding partnersuch as a substrate or another antibody. These uses can also be appliedin a therapeutic context in which treatment involves inhibiting theprotein's function. An antibody can be used, for example, to blockbinding, thus modulating (agonizing or antagonizing) the peptidesactivity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. More therapeutics methods aredescribed in detail below.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornucleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a CCAT peptide or protein of the present invention. Suchnucleic acid molecules will consist of, consist essentially of, orcomprise a nucleotide sequence that encodes one of the CCAT peptides ofthe present invention, an allelic variant thereof, or an ortholog orparalog thereof. The nucleic acid molecules and the fragments thereof ofthe present invention pertains, however, are not to be construed asencompassing fragments that may be disclosed publicly prior to thepresent invention.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in Table 1, (SEQ IDNOS:1234-2227), or any nucleic acid molecule that encodes a proteinprovided in Table 1, (SEQ ID NOS:1-1233). A nucleic acid moleculecomprises a nucleotide sequence when the nucleotide sequence is at leastpart of the final nucleotide sequence of the nucleic acid molecule. Insuch a fashion, the nucleic acid molecule can be only the nucleotidesequence or have additional nucleic acid residues, such as nucleic acidresidues that are naturally associated with it or heterologousnucleotide sequences. Such a nucleic acid molecule can have a fewadditional nucleotides or can comprise several hundred or moreadditional nucleotides. A brief description of how various types ofthese nucleic acid molecules can be readily made/isolated is providedbelow.

In Table 1, human transcript sequences are provided. As discussed below,some of the non-coding regions, particularly gene regulatory elementssuch as promoters, are useful for a variety of purposes, e.g. control ofheterologous gene expression, target for identifying gene activitymodulating compounds, and are particularly claimed as fragments of thegenomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the CCAT peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the CCAT proteins of thepresent invention that are described above. Such nucleic acid moleculesmay be naturally occurring, such as allelic variants (same locus),paralogs (different locus), and orthologs (different organism), or maybe constructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to nucleic acid molecules, cells, or organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions. Variation can occurin either or both the coding and non-coding regions. The variations canproduce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in Table 1. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in Table 1 ora fragment of this sequence. Such nucleic acid molecules can readily beidentified as being able to hybridize under moderate to stringentconditions, to the nucleotide sequence shown in the Figure sheets or afragment of the sequence. Allelic variants can readily be determined bygenetic locus of the encoding gene.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in Table 1 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in Table 1.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in Table 1. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein. The nucleic acid molecules are alsouseful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in Table 1 indicate that theCCAT of the present invention are overexpressed in colon tumor tissue.Accordingly, the probes can be used to detect the presence of, or todetermine levels of, a specific nucleic acid molecule in cells, tissues,and in organisms. The nucleic acid whose level is determined can be DNAor RNA. Accordingly, probes corresponding to the peptides describedherein can be used to assess expression and/or gene copy number in agiven cell, tissue, or organism. These uses are relevant for diagnosisof disorders involving an increase or decrease in CCAT proteinexpression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a CCAT protein, such as by measuring alevel of a CCAT-encoding nucleic acid in a sample of cells from asubject e.g., mRNA or genomic DNA, or determining if a CCAT gene hasbeen mutated. Experimental data as provided in Table 1 indicate that theCCAT of the present invention are overexpressed in colon tumor tissue.More detection and diagnosis methods are described in detail below.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate CCAT nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe CCAT gene, particularly biological and pathological processes thatare mediated by the CCAT in cells and tissues that express it.Experimental data as provided in Table 1 indicates expression in humancolon tumor tissues. The method typically includes assaying the abilityof the compound to modulate the expression of the CCAT nucleic acid andthus identifying a compound that can be used to treat a disordercharacterized by undesired CCAT nucleic acid expression. The assays canbe performed in cell-based and cell-free systems. Cell-based assaysinclude cells naturally expressing the CCAT nucleic acid or recombinantcells genetically engineered to express specific nucleic acid sequences.

The assay for CCAT nucleic acid expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the signal pathway. Further, the expression of genes thatare up- or down-regulated in response to the CCAT protein signal pathwaycan also be assayed. In this embodiment the regulatory regions of thesegenes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of CCAT gene expression can be identified in a methodwherein a cell is contacted with a candidate compound or agent and theexpression of mRNA determined. The level of expression of CCAT mRNA inthe presence of the candidate compound or agent is compared to the levelof expression of CCAT mRNA in the absence of the candidate compound oragent. The candidate compound can then be identified as a modulator ofnucleic acid expression based on this comparison and be used, forexample to treat a disorder characterized by aberrant nucleic acidexpression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound or an agent identified through drugscreening as a gene modulator to modulate CCAT nucleic acid expressionin cells and tissues that express the CCAT. Experimental data asprovided in Table 1 indicate that the CCAT of the present invention areoverexpressed in colon tumor tissue. Modulation includes bothup-regulation (i.e. activation or agonization) or down-regulation(suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for nucleic acid expression can be a smallmolecule or drug identified using the screening assays described hereinas long as the drug or small molecule inhibits the CCAT nucleic acidexpression in the cells and tissues that express the protein.Experimental data as provided in Table 1 indicates expression in humancolon tumor tissues.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds or agents on the expression oractivity of the CCAT gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in CCAT nucleic acid expression, and particularly inqualitative changes that lead to pathology. The nucleic acid moleculescan be used to detect mutations in CCAT genes and gene expressionproducts such as mRNA. The nucleic acid molecules can be used ashybridization probes to detect naturally occurring genetic mutations inthe CCAT gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the CCAT gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a CCAT protein.

Individuals carrying mutations in the CCAT gene can be detected at thenucleic acid level by a variety of techniques. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way. In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter ofwhich can be particularly useful for detecting point mutations in thegene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a CCAT gene can be directly identified, forexample, by alterations in restriction enzyme digestion patternsdetermined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and 51 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutant CCATgene and a wild-type gene can be determined by direct DNA sequencing. Avariety of automated sequencing procedures can be utilized whenperforming the diagnostic assays (Naeve, C. W., (1995) Biotechniques19:448), including sequencing by mass spectrometry (see, e.g., PCTInternational Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the CCAT gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol CCAT gene expression in cells, tissues, and organisms. A DNAantisense nucleic acid molecule is designed to be complementary to aregion of the gene involved in transcription, preventing transcriptionand hence production of CCAT protein. An antisense RNA or DNA nucleicacid molecule would hybridize to the mRNA and thus block translation ofmRNA into CCAT protein.

The nucleic acid of the present invention may also be used tospecifically suppress gene expression by methods such as RNAinterference (RNAi), which may also include cosuppression and quelling.This and antisense RNA or DNA of gene suppression are well known in theart. A review of this technique is found in Science 288:1370-1372, 2000.RNAi also operates on a post-transcriptional level and is sequencespecific, but suppresses gene expression far more efficiently thanantisense RNA. RNAi fragments, particularly double-stranded (ds) RNAi,can be also used to generate loss-of-function phenotypes.

The present invention relates to isolated RNA molecules(double-stranded; single-stranded) of from about 21 to about 25nucleotides which mediate RNAi. As used herein, about 21 to about 25 ntincludes nucleotides 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and29 nucleotides in length. The isolated RNAs of the present inventionmediate degradation of mRNA, the transcriptional product of a gene. SuchmRNA is also referred to herein as mRNA to be degraded. As used herein,the terms RNA, RNA molecule(s), RNA segment(s) and RNA fragment(s) areused interchangeably to refer to RNA that mediates RNA interference.These terms include double-stranded RNA, single-stranded RNA, isolatedRNA (partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA), as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the 21-25nt RNA or internally (at one or more nucleotides of the RNA).Nucleotides in the RNA molecules of the present invention can alsocomprise non-standard nucleotides, including non-naturally occurringnucleotides or deoxyribonucleotides. Collectively, all such altered RNAsare referred to as analogs or analogs of naturally-occurring RNA. RNA of21-25 nucleotides of the present invention need only be sufficientlysimilar to natural RNA that it has the ability to mediate RNAi. As usedherein the phrase “mediates RNAi” refers to the ability to distinguishwhich RNAs are to be degraded by the RNAi machinery or process. RNA thatmediates RNAi interacts with the RNAi machinery such that it directs thedegradation of particular mRNAs. Such RNA may include RNAs of variousstructure, including short hairpin RNA.

In one embodiment, the present invention relates to RNA molecules ofabout 21 to about 25 nucleotides that direct cleavage of specific mRNAto which their sequence corresponds. It is not necessary that there beperfect correspondence of the sequences, but the correspondence must besufficient to enable the RNA to direct RNAi cleavage of the target mRNA(Holen et al. (2005) Nucleic Acids Res. 33, 4704-4710). In a particularembodiment, the 21-25 nt RNA molecules of the present invention comprisea 3′ hydroxyl group.

The present invention relates to 21-25 nt RNAs of specific genes,produced by chemical synthesis or recombinant DNA techniques, thatmediate RNAi. As used herein, the term isolated RNA includes RNAobtained by any means, including processing or cleavage of dsRNA;production by chemical synthetic methods; and production by recombinantDNA techniques. The invention further relates to uses of the 21-25 ntRNAs, such as for therapeutic or prophylactic treatment and compositionscomprising 21-25 nt RNAs that mediate RNAi, such as pharmaceuticalcompositions comprising 21-25 nt RNAs and an appropriate carrier.

The present invention also relates to a method of mediating RNAinterference of genes of a patient. In one embodiment, RNA of about 21to about 25 nt which targets the specific mRNA to be degraded isintroduced into a patient's cells. The cells are maintained underconditions allowing degradation of the mRNA, resulting in RNA-mediatedinterference of the mRNA of the gene in the cells of the patient.Treatment of patients with cancer with the RNAi will inhibit the growthand spread of the cancer and reduce the tumor. Treatment of patientsusing RNAi can also be in combination with other anti-cancer compounds.The RNAi may be used in combination with other treatment modalities,such as chemotherapy, cryotherapy, hyperthermia, radiation therapy, andother similar treatments. In one embodiment, a chemotherapy agent wascombined with the RNAi. In another embodiment, a chemotherapy namedGemzar was used.

Treatment of cancer or tumors in patients requires introduction of theRNA into the cancer or tumor cells. RNA may be directly introduced intothe cell, or introduced extracellularly into a cavity, interstitialspace, into the circulation of a patient, or introduced orally. Methodsfor oral introduction include direct mixing of the RNA with food, aswell as engineered approaches in which a species that is used as food isengineered to express the RNA and then ingested. Physical methods ofintroducing nucleic acids, for example, injection directly into the cellor extracellular injection into the patient, may also be used. Vascularor extravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced. RNA maybe introduced into an embryonic stem cell, or another multipotent cellderived from the patient. Physical methods of introducing nucleic acidsinclude injection of a solution containing the RNA, bombardment byparticles covered by the RNA, soaking cells or tissue in a solution ofthe RNA, or electroporation of cell membranes in the presence of theRNA. A viral construct packaged into a viral particle may be used tointroduce an expression construct into the cell, with the constructexpressing RNA. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and the like. Thus the RNA may beintroduced along with components that perform one or more of thefollowing activities: enhance RNA uptake by the cell, promote annealingof the duplex strands, stabilize the annealed strands, or otherwiseincrease inhibition of the target gene. The RNAi may be used incombination with other treatment modalities, such as chemotherapy,cryotherapy, hyperthermia, radiation therapy, and the like.

The present invention may be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to tissue or patients. Preferredcomponents are the dsRNA and a vehicle that promotes introduction of thedsRNA. Such a kit may also include instructions to allow a user of thekit to practice the invention.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of CCAT nucleic acid. Accordingly,these molecules can treat a disorder characterized by abnormal orundesired CCAT nucleic acid expression. This technique involves cleavageby means of ribozymes containing nucleotide sequences complementary toone or more regions in the mRNA that attenuate the ability of the mRNAto be translated. Possible regions include coding regions andparticularly coding regions corresponding to the catalytic and otherfunctional activities of the CCAT protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in CCAT gene expression.Thus, recombinant cells, which include the patient's cells that havebeen engineered ex vivo and returned to the patient, are introduced intoan individual where the cells produce the desired CCAT protein to treatthe individual.

The invention also encompasses kits for detecting the presence of a CCATnucleic acid in a biological sample. Experimental data as provided inTable 1 indicate that the CCAT of the present invention areoverexpressed in colon tumor tissue. For example, the kit can comprisereagents such as a labeled or labelable nucleic acid or agent capable ofdetecting CCAT nucleic acid in a biological sample; means fordetermining the amount of CCAT nucleic acid in the sample; and means forcomparing the amount of CCAT nucleic acid in the sample with a standard.The compound or agent can be packaged in a suitable container. The kitcan further comprise instructions for using the kit to detect CCATprotein mRNA or DNA.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequences to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (2001).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein; increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enteroenzyme. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione 5-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001).

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 3rd. ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (2001).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as CCATs,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with CCATs, the protein can be isolated from the host cell bystandard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a CCATprotein or peptide that can be further purified to produce desiredamounts of CCAT protein or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe CCAT protein or CCAT protein fragments, such as those describedabove as well as other formats known in the art. Thus, a recombinanthost cell expressing a native CCAT protein is useful for assayingcompounds that stimulate or inhibit CCAT protein function.

Host cells are also useful for identifying CCAT protein mutants in whichthese functions are affected. If the mutants naturally occur and giverise to a pathology, host cells containing the mutations are useful toassay compounds that have a desired effect on the mutant CCAT protein(for example, stimulating or inhibiting function) which may not beindicated by their effect on the native CCAT protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a CCAT protein andidentifying and evaluating modulators of CCAT protein activity. Otherexamples of transgenic animals include non-human primates, sheep, dogs,cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the CCAT protein nucleotidesequences can be introduced as a transgene into the genome of anon-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the CCAT protein to particular cells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter Go phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyst and then transferred to pseudopregnant female foster animal.The offspring born of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, CCAT protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo CCATprotein function, including substrate interaction, the effect ofspecific mutant CCAT proteins on CCAT protein function and substrateinteraction, and the effect of chimeric CCAT proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more CCAT proteinfunctions.

Detection and Diagnosis

The present invention provides a method for detecting CCAT nucleicacids, proteins, peptides and fragments thereof that are differentiallyexpressed in colon diseases in a test sample, preferably in a biologicalsample.

The present invention further provides a method for diagnosing the colondiseases, by detecting the nucleic acids, proteins, peptides andfragments thereof. The further embodiment includes but is not limitedto, monitoring the disease prognosis (recurrance), diagnosing diseasestage, preventing the disease and treating the disease.

As used herein, a “biological sample” can be collected from tissues,blood, sera, cell lines or biological fluids such as, plasma,interstitial fluid, urine, cerebrospinal fluid, and the like, containingcells. In preferred embodiments, a biological sample comprises cells ortissues suspected of having diseases (e.g., cells obtained from abiopsy).

As used herein, a “differential level” is defined as the level of CCATprotein or nucleic acids in a test sample either above or below thelevel of the ones in control samples, wherein the level of controlsamples is obtained either from a control cell line, a normal tissue orbody fluids, or combination thereof, from a healthy subject.

As used herein, a “subject” can be a mammalian subject or non mammaliansubject, preferably, a mammalian subject. A mammalian subject can behuman or non-human, preferably human. A healthy subject is defined as asubject without detectable colon diseases or colon associated diseasesby using conventional diagnostic methods.

As used herein the “diseases” include colon diseases and colonassociated disease.

As used herein, “cancer” includes epithelial-cell related cancers, forexample pancreatic, lung, colon, prostate, ovarian, breast, bladderrenal, hepatocellular, pharyngeal and gastric cancers.

Nucleic Acid Detections

The present invention is not limited to the detection methods describedabove. Any suitable detection method that allows for the specificdetection of colon diseases cells, tissues or organs may be utilized.For example, in some embodiments, the expression of RNA corresponding toa CCAT gene is detected by hybridization to an antisense oligonucleotide(described below). In other embodiments, RNA expression is detected byhybridization assays such as Northern blots, RNase assays, reversetranscriptase PCR amplification, and the like. One preferred detectionmethod is using RT PCR by using TaqMan technology (ABI, Foster City,Calif.).

In another embodiment, the present invention provides a method fordiagnosing or detecting colon diseases in a subject comprising:determining the level of one or more CCAT nucleic acid molecules or anyfragment(s) thereof in a test sample from said subject, wherein saidCCAT nucleic acid molecule(s) comprises a sequence selected from a groupconsisting of SEQ ID NOS:1234-2227 and a combination thereof; wherein adifferential level of said CCAT nucleic acid molecule(s) relative to thelevel of said nucleic acid molecule(s) in a test sample from a healthysubject, or the level established for a healthy subject, is indicativeof colon diseases.

In another embodiment, the detecting or diagnosing method comprisesdetermining level of differential expression of 2, 4, 8, 10, 20 or morenucleic acid molecules, preferably, the nucleic acid molecules compriseor consists of a sequence selected from the group consisting of SEQ IDNOS:1234-2227 and combination thereof.

In further embodiments of the present invention, the presence ofparticular sequences in the genome of a subject is detected. Suchsequences include CCAT sequences associated with abnormal expression ofCCAT (e.g., overexpression or expression at a physiologicalinappropriate time). These sequences include polymorphisms, includingpolymorphisms in the transcribed sequence (e.g., that effect CCATprocessing and/or translation) and regulatory sequences such aspromoters, enhances, repressors, and the like. These sequences may alsoinclude polymorphisms in genes or control sequences associated withfactors that affect expression such as transcription factors, and thelike. Any suitable method for detecting and/or identifying thesesequences is within the scope of the present invention including, butnot limited to, nucleic acid sequencing, hybridization assays (e.g.,Southern blotting), single nucleotide polymorphism assays (See e.g.,U.S. Pat. No. 5,994,069, herein incorporated by reference in itsentirety), and the like.

Protein Detections

The present invention provides methods for diagnosing or detecting thedifferential presence of CCAT protein. In some embodiments (e.g., whereCCATs are overexpressed in diseased cells), CCAT proteins are detecteddirectly. In other embodiments (e.g., where the presence of a CCATs areunderexpressed), CCAT to the disease antigens are detectednon-existence.

The diagnostic methods of the present invention find utility in thediagnosis and characterization of diseases. For example, the presence ofan antibody to a specific protein may be indicative of a cancer ordisease. In addition, certain CCAT may be indicative of a specific stageor sub-type of the same cancer or disease.

The information obtained is also used to determine prognosis andappropriate course of treatment. For example, it is contemplated thatindividuals with a specific CCAT expression or stage of colon diseasesmay respond differently to a given treatment that individuals lackingthe CCAT expression. The information obtained from the diagnosticmethods of the present invention thus provides for the personalizationof diagnosis and treatment.

In one embodiment, the present invention provides a method formonitoring colon diseases treatment in a subject comprising: determiningthe level of one or more CCAT proteins or any fragment(s) or peptide(s)thereof in a test sample from said subject, wherein said CCAT protein(s)comprises a sequence selected from a group consisting of SEQ IDNOS:1-1233, SEQ ID NOS:2228-2638 and a combination thereof; wherein anlevel of said CCAT protein(s) similar to the level of said protein(s) ina test sample from a healthy subject, or the level established for ahealthy subject, is indicative of successful treatment.

In another embodiment, the present invention provides a method fordiagnosing recurrence of colon diseases following successful treatmentin a subject comprising: determining the level of one or more CCATproteins or any fragment(s) or peptide(s) thereof in a test sample fromsaid subject, wherein said CCAT protein(s) comprises a sequence selectedfrom a group consisting of SEQ ID NOS:1-1233, SEQ ID NOS:2228-2638 or acombination thereof; wherein a changed level of said CCAT protein(s)relative to the level of said protein(s) in a test sample from a healthysubject, or the level established for a healthy subject, is indicativeof recurrence of colon diseases.

In yet another embodiment, the presend invention provides a method fordiagnosing or detecting colon diseases in a subject comprising:determining the level of one or more CCAT proteins or any fragment(s) orpeptides thereof in a test sample from said subject, wherein said CCATprotein(s) comprises a sequence selected from a group consisting of SEQID NOS:1-1233, SEQ ID NOS:2228-2638 and a combination thereof; wherein adifferential level of said CCAT protein(s) relative to the level of saidprotein(s) in a test sample from a healthy subject, or the levelestablished for a healthy subject, is indicative of colon diseases.

The detecting or diagnosing method comprises determining level ofdifferential expression of 2, 4, 8, 10, 20 or more proteins, preferably,the proteins are selected from a group consisting of SEQ ID NOS:1-1233and combination thereof.

Further, the detecting or diagnosing method comprises determining levelof differential expression of 5, 10, 15, 20, 40, 60, 80, 100 or moreCCAT peptides, preferably the peptides are selected from the groupconsisting of SEQ ID NOS:2228-2638 and combination thereof.

These methods are also useful for diagnosing diseases that showdifferential protein expression. As describe earlier, normal, control orstandard values or level established from a healthy subject for proteinexpression are established by combining body fluids or tissue, cellextracts taken from a normal healthy mammalian or human subject withspecific antibodies to a protein under conditions for complex formation.Standard values for complex formation in normal and diseased tissues areestablished by various methods, often photometric means. Then complexformation as it is expressed in a subject sample is compared with thestandard values. Deviation from the normal standard and toward thediseased standard provides parameters for disease diagnosis or prognosiswhile deviation away from the diseased and toward the normal standardmay be used to evaluate treatment efficacy.

In yet another embodiment, the present invention provides a detection ordiagnostic method of CCATs by using LC/MS. The proteins from cells areprepared by methods known in the art (R. Aebersold Nature BiotechnologyVolume 21 Number 6 Jun. 2003). The differential expression of proteinsin disease and healthy samples are quantitated using Mass Spectrometryand ICAT (Isotope Coded Affinity Tag) labeling, which is known in theart. ICAT is an isotope label technique that allows for discriminationbetween two populations of proteins, such as a healthy and a diseasesample. The LC/MS spectra are collected for the labeled samples. The rawscans from the LC/MS instrument are subjected to peak detection andnoise reduction software. Filtered peak lists are then used to detect‘features’ corresponding to specific peptides from the originalsample(s). Features are characterized by their mass/charge, charge,retention time, isotope pattern and intensity.

The intensity of a peptide present in both healthy and disease samplescan be used to calculate the differential expression, or relativeabundance, of the peptide. The intensity of a peptide found exclusivelyin one sample can be used to calculate a theoretical expression ratiofor that peptide (singleton). Expression ratios are calculated for eachpeptide of each replicate of the experiment (Table 1). Thusoverexpression or under expression of CCAT protein or peptide aresimilar to the expression pattern in Table 1 in a test subject indicatesthe likelihood of having colon diseases or diseases associated withcolon.

Immunological methods for detecting and measuring complex formation as ameasure of protein expression using either specific polyclonal ormonoclonal antibodies are known in the art. Examples of such techniquesinclude enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays(RIAs), fluorescence-activated cell sorting (FACS) and antibody arrays.Such immunoassays typically involve the measurement of complex formationbetween the protein and its specific antibody. These assays and theirquantitation against purified, labeled standards are well known in theart (Ausubel, supra, unit 10.1-10.6). A two-site, monoclonal-basedimmunoassay utilizing antibodies reactive to two non-interferingepitopes is preferred, but a competitive binding assay may be employed(Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.). Moreimmunological detections are described in section below.

Antibody Detections

Antibodies are useful to detect the presence of one of the proteins orfragments thereof, peptides of the present invention in cells or tissuesto determine the pattern of expression of the protein among varioustissues in an organism and over the course of normal development.

Further, as described above, the antibodies can be used to assessexpression in disease states such as in active stages of the disease orin an individual with a predisposition toward disease related to theprotein's function. The antibodies can also be used to assess normal andaberrant subcellular localization of cells in the various tissues in anorganism.

Detection on a protein by an antibody can be facilitated by coupling(i.e., physically linking) the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials (see below). The antibodies mayalso be useful in diagnostic assays, e.g., for detecting expression ofan antigen, for example CCAT protein, peptide or fragment thereof, inspecific cells, tissues, blood, serum or body fluids.

For diagnostic applications, the antibody or its variant typically willbe labeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories:

(a) Radioisotopes, such as ³⁶S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodyvariant can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, vol 1-2, Coligen et al.,Ed., Wiley-Interscience, New York, Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody variant using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. Nos.4,275,149, 4,318,980 provides a review of some of these. The enzymegenerally catalyzes a chemical alteration of the chromogenic substratewhich can be measured using various techniques. For example, the enzymemay catalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for Use inEnzyme Immunoassay, in Methods in Enzyme. (Ed. J. Langone & H. VanVunakis), Academic press, New York, 73: 147-166 (1981).

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g. digloxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g. anti-digloxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the antibody need not belabeled, and the presence thereof can be detected using a labeledantibody, which binds to the antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The biological samples can then be tested directly for the presence ofCCAT by assays (e.g., ELISA or radioimmunoassay) and format (e.g.,microwells, dipstick (e.g., as described in International PatentPublication WO 93/03367), etc). Alternatively, proteins in the samplecan be size separated (e.g., by polyacrylamide gel electrophoresis(PAGE)), in the presence or absence of sodium dodecyl sulfate (SDS), andthe presence of CCAT detected by immunoblotting (e.g., Westernblotting). Immunoblotting techniques are generally more effective withantibodies generated against a peptide corresponding to an epitope of aprotein, and hence, are particularly suited to the present invention.

Antibody binding is detected by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay. In some embodiments, an automateddetection assay is utilized. Methods for the automation of immunoassaysare well known in the art (See e.g., U.S. Pat. Nos. 5,885,530,4,981,785, 6,159,750, and 5,358,691, each of which is hereinincorporated by reference). In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of antigens is utilized.

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample, which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, or the proteinto be detected. In a sandwich assay, the test sample to be analyzed isbound by a first antibody, which is immobilized on a solid support, andthereafter a second antibody binds to the test sample, thus forming aninsoluble three-part complex. See e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or more CCATtargets and the affinity value(Kd) is less than 1×10⁸ M.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art.

For immunohistochemistry, the disease tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin (see Example). The fixed or embedded section contains thesample are contacted with a labeled primary antibody and secondaryantibody, wherein the antibody is used to detect the CCAT proteinexpress in situ. The detailed procedure is shown in the Example.

Array:

Array technologies and quantitative PCR provide the means to explore theexpression profiles of a large number of related or unrelated genes, andproteins. When an expression profile is examined, arrays provide aplatform for examining which genes or proteins are tissue-specific,carrying out housekeeping functions, parts of a signaling cascade, orspecifically related to a particular genetic predisposition, condition,disease, or disorder. The potential application of gene or proteinexpression profiling is particularly relevant to improving diagnosis,prognosis, and treatment of disease. For example, both the sequences andthe amount of expression can be compared between tissues from subjectswith different types of colon diseases and cytologically normal healthytissue.

“Array” refers to an ordered arrangement of at least two transcripts,proteins or peptides, or antibodies on a substrate. At least one of thetranscripts, proteins, or antibodies represents a control or standard,and the other transcript, protein, or antibody is of diagnostic ortherapeutic interest. The arrangement of at least two and up to about40,000 transcripts, proteins, or antibodies on the substrate assuresthat the size and signal intensity of each labeled complex, formedbetween each transcript and at least one nucleic acid, each protein andat least one ligand or antibody, or each antibody and at least oneprotein to which the antibody specifically binds, is individuallydistinguishable.

An “expression profile” is a representation of gene expression in asample. A nucleic acid expression profile is produced using sequencing,hybridization, or amplification technologies using transcripts from asample. A protein expression profile, although time delayed, mirrors thenucleic acid expression profile and is produced using gelelectrophoresis, mass spectrometry, or an array and labeling moieties orantibodies which specifically bind the protein. The nucleic acids,proteins, or antibodies specifically binding the protein may be used insolution or attached to a substrate, and their detection is based onmethods well known in the art.

A substrate includes but not limits to, paper, nylon or other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

The invention also provides an array with a cDNA or transcript encodingCCAT proteins or peptides or fragments thereof, antibodies whichspecifically bind CCAT proteins, peptides or fragments thereof.Preferably, two or more of the nucleic acid molecules (e.g., SEQ IDNOS:1234-2227), proteins (e.g., SEQ ID NOS:1-1233) or peptides (e.g.,SEQ ID NOS:2228-2638) are immobilized on a substrate.

The present invention also provides an antibody array. Antibody arrayshave allowed the development of techniques for high-throughput screeningof recombinant antibodies. Such methods use robots to pick and gridbacteria containing antibody genes, and a filter-based ELISA to screenand identify clones that express antibody fragments. Because liquidhandling is eliminated and the clones are arrayed from master stocks,the same antibodies can be spotted multiple times and screened againstmultiple antigens simultaneously. For more information, see de Wildt etal. (2000) Nat Biotechnol 18:989-94.

The array is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), U.S.Pat. No. 5,807,522, Brown et al., all of which are incorporated hereinin their entirety by reference.

In one embodiment, a nucleic acid array or a microarray, preferablycomposed of a large number of unique, single-stranded nucleic acidsequences, usually either synthetic antisense oligonucleotides orfragments of cDNAs, fixed to a solid support. The oligonucleotides arepreferably about 6-60 nucleotides in length, more preferably 15-30nucleotides in length, and most preferably about 20-25 nucleotides inlength.

In order to produce oligonucleotides to a known sequence for an array,the gene(s) of interest (or an ORF identified from the contigs of thepresent invention) is typically examined using a computer algorithmwhich starts at the 5′ or at the 3′ end of the nucleotide sequence.Typical algorithms will then identify oligomers of defined length thatare unique to the gene, have a GC content within a range suitable forhybridization, and lack predicted secondary structure that may interferewith hybridization. In certain situations it may be appropriate to usepairs of oligonucleotides on an array. The “pairs” will be identical,except for one nucleotide that preferably is located in the center ofthe sequence. The second oligonucleotide in the pair (mismatched by one)serves as a control. The number of oligonucleotide pairs may range fromtwo to one million. The oligomers are synthesized at designated areas ona substrate using a light-directed chemical process, wherein thesubstrate may be paper, nylon or other type of membrane, filter, chip,glass slide or any other suitable solid support as described above.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference.

A gene expression profile comprises the expression of a plurality oftranscripts as measured by after hybridization with a sample. Thetranscripts of the invention may be used as elements on an array toproduce a gene expression profile. In one embodiment, the array is usedto diagnose or monitor the progression of disease. Researchers canassess and catalog the differences in gene expression between healthyand diseased tissues or cells.

For example, the transcript or probe may be labeled by standard methodsand added to a biological sample from a patient under conditions for theformation of hybridization complexes. After an incubation period, thesample is washed and the amount of label (or signal) associated withhybridization complexes, is quantified and compared with a standardvalue. If complex formation in the patient sample is significantlyaltered (higher or lower) in comparison to either a normal or diseasestandard, then differential expression indicates the presence of adisorder.

In order to provide standards for establishing differential expression,normal and disease expression profiles are established. This isaccomplished by combining a sample taken from normal subjects, eitheranimal or human or nonmammal, with a transcript under conditions forhybridization to occur. Standard hybridization complexes may bequantified by comparing the values obtained using normal subjects withvalues from an experiment in which a known amount of a purified sequenceis used. Standard values obtained in this manner may be compared withvalues obtained from samples from patients who were diagnosed with aparticular condition, disease, or disorder. Deviation from standardvalues toward those associated with a particular disorder is used todiagnose that disorder.

By analyzing changes in patterns of gene expression, disease can bediagnosed at earlier stages before the patient is symptomatic. Theinvention can be used to formulate a prognosis and to design a treatmentregimen. The invention can also be used to monitor the efficacy oftreatment. For treatments with known side effects, the array is employedto improve the treatment regimen. A dosage is established that causes achange in genetic expression patterns indicative of successfultreatment. Expression patterns associated with the onset of undesirableside effects are avoided.

In another embodiment, animal models which mimic a human disease can beused to characterize expression profiles associated with a particularcondition, disease, or disorder; or treatment of the condition, disease,or disorder. Novel treatment regimens may be tested in these animalmodels using arrays to establish and then follow expression profilesover time. In addition, arrays may be used with cell cultures or tissuesremoved from animal models to rapidly screen large numbers of candidatedrug molecules, looking for ones that produce an expression profilesimilar to those of known therapeutic drugs, with the expectation thatmolecules with the same expression profile will likely have similartherapeutic effects. Thus, the invention provides the means to rapidlydetermine the molecular mode of action of a drug.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies or in clinical trials orto monitor the treatment of an individual patient. Once the presence ofa condition is established and a treatment protocol is initiated,diagnostic assays may be repeated on a regular basis to determine if thelevel of expression in the patient begins to approximate that which isobserved in a normal subject. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to years.

Treatment

The following terms, as used in the present specification and claims,are intended to have the meaning as defined below, unless indicatedotherwise.

“Treat,” “treating” or “treatment” of a disease includes: (1) inhibitingthe disease, i.e., arresting or reducing the development of the diseaseor its clinical symptoms, or (2) relieving the disease, i.e., causingregression of the disease or its clinical symptoms.

A “therapeutically effective amount” means the amount of an agent that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” will vary depending on the agent, the disease and its severityand the age, weight, etc., of the subject to be treated.

A “Colon or colorectal disease” includes but not limited to coloncancer, colon tumor, diverticulosis, diverticulitis, Crohn's disease,ulcerative colitis, irritable bowel syndrome, inflammatory boweldisease, hemorrhoids, and anal fissure.

A “cancer” is epithelial-cell related cancers include but not limited topancreatic, lung, colon, prostate, ovarian, breast, bladder renal,hepatocellular, pharyngeal and gastric cancer.

The present invention provides an application of treatment by usingantibody, immunogenic peptides as well as other CCAT agonists orantagonists.

CCATs are proteins differentially expressed in the colon diseases celllines or tissues. The proteins are either cell surface proteins,cytosolic proteins, or secreted proteins (see the list in Table 1).These proteins are associated with the diseases especially colondiseases, particularly colon cancer; thus, they serve as candidatetargets for the treatment of the diseases.

In one embodiment, when decreased expression or activity of the proteinis desired, an inhibitor, antagonist, antibody and the like or apharmaceutical agent containing one or more of these molecules may bedelivered. Such delivery may be effected by methods well known in theart and may include delivery by an antibody specifically targeted to theprotein. Neutralizing antibodies, which inhibit dimer formation, aregenerally preferred for therapeutic use.

In another embodiment, when increased expression or activity of theprotein is desired, the protein, an agonist, an enhancer and the like ora pharmaceutical agent containing one or more of these molecules may bedelivered. Such delivery may be effected by methods well known in theart and may include delivery of a pharmaceutical agent by an antibodyspecifically targeted to the protein.

Any of the transcripts, complementary molecules, or fragments thereof,proteins or portions thereof, vectors delivering these nucleic acidmolecules or expressing the proteins, and their ligands may beadministered in combination with other therapeutic agents. Selection ofthe agents for use in combination therapy may be made by one of ordinaryskill in the art according to conventional pharmaceutical principles. Acombination of therapeutic agents may act synergistically to affecttreatment of a particular disorder at a lower dosage of each agent.

Antibody Therapy

The antibody of the present invention can be used for therapeuticreason. It is contemplated that the antibody of the present inventionmay be used to treat a mammal, preferably human with colon diseases.

In general, the antibodies are also useful for inhibiting proteinfunction, for example, blocking the binding of the CCAT protein orpeptide to a binding partner such as a substrate. These uses can also beapplied in a therapeutic context in which treatment involves inhibitingthe protein's function. An antibody can be used, for example, to blockbinding, thus modulating (agonizing or antagonizing) the peptidesactivity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated within a cell or cell membrane. The function blocking assaysare provided in detail in the Examples.

The antibodies of present invention can also be used as means ofenhancing the immune response. The antibodies can be administered inamounts similar to those used for other therapeutic administrations ofantibody. For example, pooled gamma globulin is administered at a rangeof about 1 mg to about 100 mg per patient. Thus, antibodies reactivewith the protein or peptides of CCAT can be passively administered aloneor in conjunction with other anti-cancer therapies to a mammalafflicted-with colon diseases or cancer. Examples of anti-cancertherapies include, but are not limited to, chemotherapy, radiationtherapy, adoptive immunotherapy therapy with TIL (Tumor InfiltrationLymphocytes).

The selection of an antibody subclass for therapy will depend upon thenature of the disease tumor antigen. For example, an IgM may bepreferred in situations where the antigen is highly specific for thediseased target and rarely occurs on normal cells. However, where thedisease-associated antigen is also expressed in normal tissues, althoughat much lower levels, the IgG subclass may be preferred for thefollowing reason: since the binding of at least two IgG molecules inclose proximity is required to activate complement, less complementmediated damage may occur in the normal tissues which express smalleramounts of the antigen and, therefore, bind fewer IgG antibodymolecules. Furthermore, IgG molecules by being smaller may be more ablethan IgM molecules to localize to the diseased tissue.

The mechanism for antibody therapy is that the therapeutic antibodyrecognizes a cell surface protein, cytosolic protein, or a secretedprotein that is overexpressed in diseased cells. By NK cell orcomplement activation, conjugation of the antibody with an immunotoxinor radiolabel, the interaction can abrogate ligand/respecter interactionor activation of apoptosis.

The potential mechanisms of antibody-mediated cytotoxicity of diseasedcells are phagocyte (antibody dependent cellular cytotoxicity (ADCC))(see Example), complement (Complement-mediated cytotoxicity (CMC)) (seeExample), naked antibody (receptor cross-linking apoptosis and growthfactor inhibition), or targeted payload labeled with radionuclide orimmunotoxins or immunochemotherapeutics.

In one embodiment, the antibody is administered to a nonhuman mammal forthe purposes of obtaining preclinical data, for example. Exemplarynonhuman mammals to be treated include nonhuman primates, dogs, cats,rodents and other mammals in which preclinical studies are performed.Such mammals may be established animal models for a disease to betreated with the antibody or may be used to study toxicity of theantibody of interest. In each of these embodiments, dose escalationstudies may be performed on the mammal.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody variant issuitably administered by pulse infusion, particularly with decliningdoses of the antibody variant. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

For the prevention or treatment of a disease, the appropriate dosage ofthe antibody will depend on the type of disease to be treated, theseverity and the course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician.

Depending on the type and severity of the disease, about 1 .mu.g/kg to150 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

The antibody composition will be formulated, dosed and administered in amanner consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

The therapeutically effective amount of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question.

Antibodies of the present invention may also be used as therapeuticreagents, to diminish or eliminate cancer or tumors. For example, theantibodies may be used on their own (for instance, to inhibitmetastases) or coupled to one or more therapeutic agents. Suitableagents in this regard include radionuclides, differentiation inducers,drugs, toxins, and derivatives thereof. Preferred radionuclides include⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and ²¹²Bi. Preferred drugsinclude methotrexate, and pyrimidine and purine analogs. Preferreddifferentiation inducers include phorbol esters and butyric acid.Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin,gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviralprotein

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be affected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g. U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways as described above.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation with a carrier.

The formulations of the present invention, both for veterinary and forhuman use, comprise an immunogen as described above, together with oneor more pharmaceutically acceptable carriers and, optionally, othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethod well-known in the pharmaceutical art.

Suitable pharmaceutical carriers include proteins such as albumins(e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides andpolysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784, toShih et al.), or water. A carrier may also bear an agent by noncovalentbonding or by encapsulation, such as within a liposome vesicle (e.g.,U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers specific forradionuclide agents include radiohalogenated small molecules andchelating compounds. For example, U.S. Pat. No. 4,735,792 disclosesrepresentative radiohalogenated small molecules and their synthesis. Aradionuclide chelate may be formed from chelating compounds that includethose containing nitrogen and sulfur atoms as the donor atoms forbinding the metal, metal oxide, radionuclide. For example, U.S. Pat. No.4,673,562, to Davison et al. discloses representative chelatingcompounds and their synthesis.

All methods include the step of bringing into association the activeingredient with the carrier, which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions, which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1-2.0M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids, which may be usedeither on their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11-10,000 parts by weight per part byweight of immunogen. If two or more stabilizers are to be used, theirtotal amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. Informulating the antibody of the present invention, anti-adsorption agentmay be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate the CCATantibody into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetatecopolymers. Alternatively, instead of incorporating these agents intopolymeric particles, it is possible to entrap these materials inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

The therapeutic antibody may be supplied in the form of a kit, alone, orin the form of a pharmaceutical composition as described above.

Other Immunotherapy

The CCAT proteins or peptides or fragments thereof of this invention arealso intended for use in producing antiserum designed for pre- orpost-disease prophylaxis. Here the protein, peptides or fragmentthereof, is formulated with a suitable adjuvant and administered byinjection to human volunteers, according to known methods for producinghuman antisera. Antibody response to the injected proteins is monitored,during a several-week period following immunization, by periodic serumsampling to detect the presence of antiserum antibodies, using animmunoassay as described herein.

The antiserum from immunized individuals may be administered as aprophylactic measure for individuals who are at risk of developing colondiseases or cancer. The antiserum is also useful in treating anindividual afflicted with colon diseases or cancer for post-diseaseprophylaxis.

Alternatively, peptides derived form the CCAT protein sequence may bemodified to increase their immunogenicity by enhancing binding of thepeptide to the MHC molecules in which the peptide is presented. Thepeptide or modified peptide may be conjugated to a carrier molecule toenhance the antigenicity of the peptide. Examples of carrier molecules,include, but are not limited to, human albumin, bovine albumin,lipoprotein and keyhole limpet hemo-cyanin (“Basic and ClinicalImmunology” (1991) Stites, D. P. and Ten A. I. (eds) Appleton and Lange,Norwalk Conn., San Mateo, Calif.).

An “immunogenic peptide” is a peptide, which comprises anallele-specific motif such that the peptide will bind the MHC allele(HLA in human) and be capable of inducing a CTL (cytoxic T-lymphocytes)response. Thus, immunogenic peptides are capable of binding to anappropriate class I or II MHC molecule and inducing a cytotoxic T cellor T helper cell response against the antigen from which the immunogenicpeptide is derived.

Alternatively, amino acid sequence variants of the peptide can beprepared by mutations in the DNA, which encodes the peptide, or bypeptide synthesis.

At the genetic level, these variants ordinarily are prepared bysite-directed mutagenesis of nucleotides in the DNA encoding the peptidemolecule, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. The variants typicallyexhibit the same qualitative biological activity as the nonvariantpeptide.

T-lymphocytes recognize antigen in association with Class I or Class IIMHC molecules in the form of a peptide fragment bound to an MHCmolecule. The degree of peptide binding to a given MHC allele is basedon amino acids at particular positions within the peptide (Parker et al.(1992) Journal of Immunology 149:3580; Kubo, et al. (1994) Journal ofImmunology 52:3913-3924; Ruppert J. et al. (1993) Cell 74:929-937; Falket al. (1991) Nature 351:290-296). The peptides of the present inventionare useful as an epitope for immunogenic response (see more detaileddescription below).

In human, MHC is called HLA, wherein class I molecules are encoded bythe HLA-A, B, and C loci. HLA-A and B antigens are expressed at the cellsurface at approximately equal densities, whereas the expression ofHLA-C is significantly lower (about 10-fold lower). Each of these locihas a number of alleles. MHC class II molecules are encoded by threepairs of MHC II alpha- and beta-chain genes, called HLA DR, -DP, and -DQin human. In many haplotypes the HLA-DR cluster contains an extrabeta-chain gene whose product can pair with the DR alpha chain. Each MHCclass I and II molecule binds a different rage of peptides. The presentof several loci means that any one individual is equipped to present amuch broader ranger of different peptides than if only one MHC proteinof each class were expressed at the cell surface. The peptide bindingmotifs of the present invention are designed to be specific for eachallelic subtype.

The peptides of the present invention are used for treatment of thecolon diseases. Treatment involves administration of the protectivecomposition after the appearance of the disease.

The present invention is also applied to prevent and suppress thedisease. It is not always possible to distinguish between “preventing”and “suppressing” since the ultimate inductive event or events may beunknown, latent, or the patient is not ascertained until well after theoccurrence of the event or events. Therefore, it is common to use theterm “prophylaxis” as distinct from “treatment” to encompass both“preventing” and “suppressing” as defined herein. The term “protection,”as used herein, is meant to include “prophylaxis.”

The peptides are used for treating T cell-mediated pathology. The term“T cell-mediated pathology” refers to any condition in which aninappropriate T cell response is a component of the pathology. The termis intended to encompass both T cell mediated colon diseases anddiseases resulting from unregulated clonal T cell replication.

Therefore, the present invention relates to peptides or modifiedpeptides derived from the protein sequences of the CCAT proteins thatdifferentially expressed in the colon diseases. By way of example,modification may include substitution, deletion or addition of an aminoacid in the given immunogenic peptide sequence or mutation of existingamino acids within the given immunogenic peptide sequence, orderivatization of existing amino acids within the given immunogenicpeptide sequence. Any amino acid comprising the immunogenic peptidesequence may be modified in accordance with this invention. In oneaspect, at least one amino acid is substituted or replaced within thegiven immunogenic peptide sequence. Any amino acid may be used tosubstitute or replace a given amino acid within the immunogenic peptidesequence. Modified peptides are intended to include any immunogenicpeptide obtained from differentially expressed proteins, which has beenmodified and exhibits enhanced binding to the MHC molecule with which itassociates when presented to the T-cell. These modified peptides may besynthetically or recombinantly produced by conventional methods.

In another embodiment, the peptides of the present invention comprise,or consisting sequences of about 5-8, 8-10, 10-15 or 15-30 amino acidswhich are immunogenic, that is, capable of inducing an immune responsewhen injected into a subject.

The recombinant or natural protein, peptides, or fragment thereof ofCCAT, or modified peptides, may be used as a vaccine eitherprophylactically or therapeutically. When provided prophylactically thevaccine is provided in advance of any evidence of colon diseases,particularly, cancer. The prophylactic administration of the colondiseases vaccine should serve to prevent or attenuate colon diseases,preferably cancer, in a mammal.

Preparation of vaccine is using recombinant protein or peptideexpression vectors comprising all or part of nucleic acid sequence ofCCAT proteins encoding peptides. Examples of vectors that may be used inthe aforementioned vaccines include, but are not limited to, defectiveretroviral vectors, adenoviral vectors vaccinia viral vectors, fowl poxviral vectors, or other viral vectors (Mulligan, R. C., (1993) Science260:926-932). The viral vectors carrying all or part of nucleic sequenceof SEQ ID NOS:1234-2227 can be introduced into a mammal either prior toany evidence of colon diseases or to mediate regression of the diseasein a mammal afflicted with colon diseases. Examples of methods foradministering the viral vector into the mammals include, but are notlimited to, exposure of cells to the virus ex vivo, or injection of theretrovirus or a producer cell line of the virus into the affected tissueor intravenous administration of the virus. Alternatively the viralvector carrying all or part of the CCAT nucleic acid sequence thatencode peptides may be administered locally by direct injection into thecancer lesion or topical application in a pharmaceutically acceptablecarrier. The quantity of viral vector, carrying all or part of the CCATnucleic acid sequence, to be administered is based on the titer of virusparticles. A preferred range of the immunogen to be administered may beabout 106 to about 1011 virus particles per mammal, preferably a human.After immunization the efficacy of the vaccine can be assessed byproduction of antibodies or immune cells that recognize the antigen, asassessed by specific lytic activity or specific cytokine production orby tumor regression. One skilled in the art would know the conventionalmethods to assess the aforementioned parameters. If the mammal to beimmunized is already afflicted with cancer, the vaccine can beadministered in conjunction with other therapeutic treatments. Examplesof other therapeutic treatments includes, but are not limited to,adoptive T cell immunotherapy, coadministration of cytokines or othertherapeutic drugs for cancer.

Alternatively all or parts thereof of a substantially or partiallypurified the CCAT protein or their peptides may be administered as avaccine in a pharmaceutically acceptable carrier. Ranges of the proteinthat may be administered are about 0.001 to about 100 mg per patient,preferred doses are about 0.01 to about 100 mg per patient. In apreferred embodiment, the peptides or modified peptides thereof isadministered therapeutically or prophylactically to a mammal in need ofsuch treatment. The peptide may be synthetically or recombinantlyproduced. Immunization is repeated as necessary, until a sufficienttiter of anti-immunogen antibody or immune cells has been obtained.

In yet another alternative embodiment a viral vector, such as aretroviral vector, can be introduced into mammalian cells. Examples ofmammalian cells into which the retroviral vector can be introducedinclude, but are not limited to, primary mammalian cultures orcontinuous mammalian cultures, COS cells, NIH3T3, or 293 cells (ATTC#CRL 1573), dendritic cells. The means by which the vector carrying thegene may be introduced into a cell includes, but is not limited to,microinjection, electroporation, transfection or transfection using DEAEdextran, lipofection, calcium phosphate or other procedures known to oneskilled in the art (Sambrook et al. (EDS) (2001) in “Molecular Cloning.A laboratory manual”, Cold Spring Harbor Press Plainview, N.Y.).

The vaccine formulation of the present invention comprises an immunogenthat induces an immune response directed against the cancer associatedantigens such as the CCATs, and in nonhuman primates and finally inhumans. The safety of the immunization procedures is determined bylooking for the effect of immunization on the general health of theimmunized animal (weight change, fever, appetite behavior etc.) andlooking for pathological changes on autopsies. After initial testing inanimals, cancer patients can be tested. Conventional methods would beused to evaluate the immune response of the patient to determine theefficiency of the vaccine.

Measurement of candidate disease tumor antigen or vaccine expression inpatients is the first step of the present invention. Subsequent stepswill focus on measuring immune responses to these candidate antigens orvaccine. Sera from disease patients, particularly cancer patients, andhealthy donors will be screened for antibodies to the candidate antigensas well as for levels of circulating tumor derived antigens. antigen.The vaccine formulations may be evaluated first in animal models,initially rodents

In one embodiment mammals, preferably human, at high risk for colondiseases, particularly cancer, are prophylactically treated with thevaccines of this invention. Examples of such mammals include, but arenot limited to, humans with a family history of colon diseases, humanswith a history of colon diseases, particular cancer, or humans afflictedwith colon cancer previously resected and therefore at risk forreoccurrence. When provided therapeutically, the vaccine is provided toenhance the patient's own immune response to the diseased antigenpresent on the colon diseases or advanced stage of colon diseases. Thevaccine, which acts as an immunogen, may be a cell, cell lysate fromcells transfected with a recombinant expression vector, cell lysatesfrom cells transfected with a recombinant expression vector, or aculture supernatant containing the expressed protein. Alternatively, theimmunogen is a partially or substantially purified recombinant protein,peptide or analog thereof or modified peptides or analogs thereof. Theproteins or peptides may be conjugated with lipoprotein or administeredin liposomal form or with adjuvant.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation. The formulationsof the present invention are described in the previous section.

Vaccination can be conducted by conventional methods. For example, theimmunogen can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. Further, the immunogen may or may notbe bound to a carrier to make the protein immunogenic. Examples of suchcarrier molecules include but are not limited to bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like.The immunogen also may be coupled with lipoproteins or administered inliposomal form or with adjuvants. The immunogen can be administered byany route-appropriate for antibody production such as intravenous,intraperitoneal, intramuscular, subcutaneous, and the like. Theimmunogen may be administered once or at periodic intervals until asignificant titer of anti-CCAT immune cells or anti-CCAT antibody isproduced. The presence of anti-CCAT immune cells may be assessed bymeasuring the frequency of precursor CTL (cytoxic T-lymphocytes) againstCCAT antigen prior to and after immunization by a CTL precursor analysisassay (Coulie, P. et al., (1992) International Journal Of Cancer50:289-297). The antibody may be detected in the serum using theimmunoassay described above.

The safety of the immunization procedures is determined by looking forthe effect of immunization on the general health of the immunized animal(weight change, fever, appetite behavior etc.) and looking forpathological changes on autopsies. After initial testing in animals,colon diseases patients can be tested. Conventional methods would beused to evaluate the immune response of the patient to determine theefficiency of the vaccine.

In yet another embodiment of this invention all, part, or parts of theCCAT proteins or peptides or fragments thereof, or modified peptides,may be exposed to dendritic cells cultured in vitro. The cultureddendritic cells provide a means of producing T-cell dependent antigenscomprised of dendritic cell modified antigen or dendritic cells pulsedwith antigen, in which the antigen is processed and expressed on theantigen activated dendritic cell. The CCAT antigen activated dendriticcells or processed dendritic cell antigens may be used as immunogens forvaccines or for the treatment of colon diseases, particularly coloncancer. The dendritic cells should be exposed to antigen for sufficienttime to allow the antigens to be internalized and presented on thedendritic cells surface. The resulting dendritic cells or the dendriticcell process antigens can than be administered to an individual in needof therapy. Such methods are described in Steinman et al. (WO93/208185)and in Banchereau et al. (EPO Application 0563485A1).

In yet another aspect of this invention T-cells isolated fromindividuals can be exposed to the CCAT proteins, peptides or fragmentthereof, or modified peptides in vitro and then administered to apatient in need of such treatment in a therapeutically effective amount.Examples of where T-lymphocytes can be isolated include but are notlimited to, peripheral blood cells lymphocytes (PBL), lymph nodes, ortumor infiltrating lymphocytes (TIL). Such lymphocytes can be isolatedfrom the individual to be treated or from a donor by methods known inthe art and cultured in vitro (Kawakami, Y. et al. (1989) J. Immunol.142: 2453-3461). Lymphocytes are cultured in media such as RPMI or RPMI1640 or AIM V for 1-10 weeks. Viability is assessed by trypan blue dyeexclusion assay. Examples of how these sensitized T-cells can beadministered to the mammal include but are not limited to,intravenously, intraperitoneally or intralesionally. Parameters that maybe assessed to determine the efficacy of these sensitized T-lymphocytesinclude, but are not limited to, production of immune cells in themammal being treated or tumor regression. Conventional methods are usedto assess these parameters. Such treatment can be given in conjunctionwith cytokines or gene modified cells (Rosenberg, S. A. et al. (1992)Human Gene Therapy, 3: 75-90; Rosenberg, S. A. et al. (1992) Human GeneTherapy, 3: 57-73).

The present invention is further described by the following example. Theexample is provided solely to illustrate the invention by reference tospecific embodiments. This exemplification, while illustrating certainaspects of the invention, does not offer the limitations or circumscribethe scope of the disclosed invention.

All examples outlined here were carried out using standard techniques,which are well known and routine to those of skill in the art. Routinemolecular biology techniques of the following example can be carried outas described in standard laboratory manuals, such as Sambrook et al.,Molecular Cloning: A laboratory Manual, 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (2001).

WORKING EXAMPLES 1. Colon Tissues and Cell Lines Colon Tissues TissueProcessing

All tissues were procured as fresh specimens. Tissues were collected asremnant tissues following surgical resection of colorectal tissues.Remnant tissues were supplied following processing for pathologicaldiagnosis according to proper standards of patient care. Procurement ofall tissues was performed in an anonymised manner in strict compliancewith Federal mandated ethical and legal guidelines (HIPAA) and inaccordance with clinical institution ethical review board as well as theinternal institutional review board. Tissues were transported on ice inice-cold transport buffer by courier for processing.

i) Enrichment of Epithelial Cells from Normal Colorectal Mucosa

Normal colorectal tissue was transferred from the transport vessel to asterile dish containing 25 ml of ice-cold transport buffer. The tissuewas measured, weighed and photographed. The tissue was dissected toisolate colorectal mucosa which was transferred to a fresh dishcontaining 25 ml ice-cold Hanks buffered saline solution. The tissuesection was washed by vigorous shaking and the HBSS replaced. This wasrepeated 2 further times or until all visible mucus was removed. Mucosawas measured, weighed and diced into 1 mm2 sections. The tissuessections were transferred to a 50 ml polypropylene centrifuge tubecontaining 50 ml of A52 media (Biosource) supplemented with 2 mML-glutamine and 1.5 mg/ml dispase (Roche Biochemicals). The digest wasincubated for 1 h at 37° C. with frequent agitation. Following theincubation, the suspension was poured through a 40-mesh cell sievesituated in the base of a 15 cm culture dish. The filtrate was dilutedto 50 ml using A52 media supplemented with 2 mM L-glutamine and passedthrough a 200-mesh cell sieve. The filtrate was collected into a 50 mlpolypropylene centrifuge tube and the suspension was triturated severaltimes followed by vortexing for 2 min at setting 6. The density andviability of nucleated cells was determined by flow cytometry usingpropidium iodide as a negative stain for viability (Guava system).Erythrocytes were lysed using a standard ammonium chloride lysisprotocol with incubation at room temperature for 10s. Cells wereharvested by centrifugation at 500 g for 5 min at 4° C. The cell pelletwas resuspended in 50 ml of ice-cold HBSS and recentrifuged. The finalcell pellet was resuspended in 3 ml of ice-cold HBSS supplemented with0.1% BSA and 0.25M EDTA. Cell density and viability were estimated usingthe Guava system and the density adjusted to 1×10⁷ cells per ml.Epithelial cells were stained with a FITC-labeled anti-EpCAM murinemonoclonal antibody and enriched by cell sorting using flow cytometry.

ii) Enrichment of Tumor Cells from Colorectal Tumor Tissue

Colorectal tumor tissue was transferred from the transport vessel to asterile dish containing 25 ml of ice-cold transport buffer. The tissuewas measured, weighed and photographed. The tissue was dissected toremove necrotic and fibrotic tissue plaques and the tumour tissuetransferred to a fresh dish containing 25 ml ice-cold Hanks bufferedsaline solution. The tissue section was washed by vigorous shaking andthe HBSS replaced. This was repeated 2 further times or until allvisible mucus was removed. Tumour tissue was measured, weighed andextensively diced. The tissues slurry was transferred to a 50 mlpolypropylene centrifuge tube containing 50 ml of A52 media (Biosource)supplemented with 2 mM L-glutamine and 1.5 mg/ml dispase (RocheBiochemicals). The digest was incubated for 1 h at 37° C. with frequentagitation. Following the incubation, the suspension was poured through a40-mesh cell sieve situated in the base of a 15 cm culture dish. Thefiltrate was diluted to 50 ml using A52 media supplemented with 2 mML-glutamine and passed through a 200-mesh cell sieve. The filtrate wascollected into a 50 ml polypropylene centrifuge tube and the suspensionwas triturated several times followed by vortexing for 2 min at setting6. The density and viability of nucleated cells was determined by flowcytometry using propidium iodide as a negative stain for viability(Guava system). Erythrocytes were lysed using a standard ammoniumchloride lysis protocol with incubation at room temperature for 10s.Cells were harvested by centrifugation at 500 g for 5 min at 4° C. Thecell pellet was resuspended in 50 ml of ice-cold HBSS and recentrifuged.The final cell pellet was resuspended in 3 ml of ice-cold HBSSsupplemented with 0.1% BSA and 0.25M EDTA. Cell density and viabilitywere estimated using the Guava system and the density adjusted to 1×10⁷cells per ml. Epithelial cells were stained with a FITC-labeledanti-EpCAM murine monoclonal antibody and enriched by cell sorting usingflow cytometry.

iii) Enrichment of Cell Surface Proteins from Sorted Epithelial andTumor Cells

Sorted cells were centrifuged at 500 g at 4° C. for 5 min andresuspended in 50 ml of ice-cold DPBS. The cell suspension was washed by2 further cycles of centrifugation 500 g at 4° C. for 5 min andresuspension of the cell pellet in 50 ml of ice-cold DPBS. Finally, thecell pellet was resuspended in 9.5 ml of ice-cold DPBS and sodiummetaperiodate added to a final concentration of 1 mM. The cellsuspension was incubated on ice for 10 min with frequent agitation inthe dark. Cells were centrifuged at 500 g at 4° C. for 5 min andresuspended in 50 ml of ice-cold DPBS. The cell suspension was washed by2 further cycles of centrifugation 500 g at 4° C. for 5 min andresuspension of the cell pellet in 50 ml of ice-cold DPBS. Finally, thecell pellet was resuspended in lysis buffer (1% SDS [w/v]; 0.1M HEPES;10 mM MgCl₂; 0.1% Non ionic detergent P40; 10 μl/ml protease inhibitorcocktail [P8340, Sigma]) and homogenisation performed by passage oflysate through a 18G syringe needle 10 times. Protein concentrationswere assayed relative to a Bovine serum albumin standard by a modifiedLowry assay (DC assay, BioRAD) and 1 mg of total cellular proteintransferred to a fresh tube and diluted to 1 mg/ml in acetate buffer(0.1M, pH 5.0).

Colon Cancer Cell Line Model System:

The model system employed here involves the use of Caco2 as a “normal”reference (i.e., control) to which cell surface expression in tumorderived cell lines is compared. These differentials or candidates arethen validated in tissues, colon cancer and normal colon, to confirmthat they are differentially expressed between these tissues as well aswithin the cell line model system.

Colon Cancer Cell Line Culture

Cell lines were grown in a culturing medium that is supplemented asnecessary with growth factors and serum, in accordance with the AmericanType Culture Collection (ATCC) (Mannassas, Va.) guidelines for eachparticular cell line (the ATCC catalog number for each cell line isindicated in Table 1). Cultures were established from frozen stocks inwhich the cells were suspended in a freezing medium (cell culture mediumwith 10% DMSO [v/v]) and flash frozen in liquid nitrogen. Frozen stocksprepared in this way were stored in the liquid nitrogen vapour. Cellcultures were established by rapidly thawing frozen stocks at 37° C.Thawed stock cultures were slowly transferred to a culture vesselcontaining a large volume of culture medium that was supplemented. Formaintenance of culture, cells were seeded at 1×10⁵ cells/per ml inmedium and incubated at 37° C. until confluence of cells in the culturevessel exceeds 50% by area. At this time, cells were harvested from theculture vessel using enzymes or EDTA where necessary. The density ofharvested, viable cells was estimated by hemocytometry and the culturereseeded as above. A passage of this nature was repeated no more than 25times at which point the culture was destroyed and reestablished fromfrozen stocks as described above.

For the analyses of cell surface protein expression in cultured celllines, cells were grown as described above. At a period 24 h prior tothe experiment, the cell line was passaged as described above. Thisyielded cell densities that were <50% confluent and growingexponentially. Typically, triplicate analyses of differential expressionwere performed for each line relative to Caco2 for the purpose ofidentifying statistically significant reproducible differentiallyexpressed proteins.

2. Cloning and Expression of Target Proteins

cDNA Retrieval

Peptide sequences were searched by BlastP against the Celera DiscoverySystem (CDS) and public database to identify the correspondingfull-length open reading frames (ORFs). Each ORF sequence was thensearched by BlastN against the Celera in-house human cDNA clonecollection. For each sequence of interest, up to three clones are pulledand streaked onto LB/Ampicillin (100 ug/ml) plates. Plasmid DNA isisolated using Qiagen spin mini-prep kit and verified by restrictiondigest. Subsequently, the isolated plasmid DNA is sequence verifiedagainst the ORF reference sequence. Sequencing reactions are carried outusing Applied Biosystems BigDye Terminator kit followed by ethanolprecipitation. Sequence data is collected using the Applied Biosystems3100 Genetic Analyzer and analyzed by alignment to the referencesequence using the Clone Manager alignment tool.

PCR

PCR primers are designed to amplify the full-length ORF as well as anyregions of the ORF that are interest for expression (antigenic orhydrophilic regions as determined by the Clone Manager sequence analysistool). Primers also contain 5′ and 3′ overhangs to facilitate cloning(see below). PCR reactions contain 2.5 units Platinum Taq DNA PolymeraseHigh Fidelity (Invitrogen), 50 ng cDNA plasmid template, 1 uM forwardand reverse primers, 800 uM dNTP cocktail (Applied Biosystems) and 2 mMMgSO4. After 20-30 cycles (94° C. for 30 seconds, 55° C. for 1 minutesand 73° C. for 2 minutes), product is verified and quantitated byagarose gel electrophoresis.

Construction of Entry Clones

PCR products are cloned into an entry vector for use with the Gatewayrecombination based cloning system (Invitrogen). These vectors includepDonr221, pDonr201, pEntr/D-TOPO or pEntr/SD/D-TOPO and are used asdescribed in the cloning methods below.

TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO

For cloning using this method, the forward PCR primer contained a 5′overhang containing the sequence “CACC”. PCR products are generated asdescribed above and cloned into the entry vector using the InvitrogenTOPO cloning kit. Reactions are typically carried out at roomtemperature for 10 minutes and subsequently transformed into TOP10chemically competent cells (Invitrogen, Calif.). Candidate clones arepicked, plasmid DNA is prepared using Qiagen spin mini-prep kit andscreened using restriction digest. Inserts are subsequently sequenceverified as described above.

Gateway Cloning into pDonr201 or pDonr221

For cloning using this method, PCR primers contained the followingoverhangs:

Forward 5′ overhang:

5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3′

Reverse 5′ overhang:

5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′

PCR products are generated as described above. ORFs are recombined intothe entry vector using the Invitrogen Gateway BP Clonase enzyme mix.Reactions are typically carried out at 25° C. for 1 hour, treated withProteinase K at 37° C. for 10 minutes and transformed into LibraryEfficiency DH5a chemically competent cells (Invitrogen, Calif.).Candidate clones are picked, plasmid DNA is prepared using Qiagen spinmini-prep kit and screened using restriction digest. Inserts aresubsequently sequence verified as described above.

Construction of Expression Clones

ORFs are transferred from the entry construct into a series ofexpression vectors using the Gateway LR Clonase enzyme mix. Reactionsare typically carried out for 1 hour at 25° C., treated with ProteinaseK at 37° C. for 10 minutes and subsequently transformed into LibraryEfficiency DH5a chemically competent cells (Invitrogen). Candidateclones are picked, plasmid DNA is prepared using Qiagen spin mini-prepkit and screened using restriction digest. Expression vectors includebut are not limited to pDest14, pDest15, pDest17, pDest8, pDest10 andpDest20. These vectors allow expression in systems such as E. coli andrecombinant baculovirus. Other vectors not listed here allow expressionin yeast, mammalian cells, or in vitro.

Expression of Recombinant Proteins in E. coli

Constructs are transformed into one or more of the following hoststrains: BL21 SI, BL21 AI, (Invitrogen); Origami B (DE3), Origami B(DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Rosetta-Gami (DE3),Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B (DE3) pLysS (Novagen). Thetransformants are grown in LB with or without NaCl and with appropriateantibiotics, at temperatures in the range of 20-37° C., with aeration.Expression is induced with the addition of IPTG (0.03-0.3 mM) or NaCl(75-300 mM) when the cells are in mid-log growth. Growth is continuedfor one to 24 hours post-induction. Cells are harvested bycentrifugation in a Sorvall RC-3C centrifuge in a H6000A rotor for 10minutes at 3000 rpm, at 4° C. Cell pellets are stored at −80° C.

Expression of Recombinant Proteins Using Baculovirus

Recombinant proteins are expressed using baculovirus in Sf21 fall armyworm ovarian cells. Recombinant baculoviruses are prepared using theBac-to-Bac system (Invitrogen) per the manufacturer's instructions.Proteins are expressed on the large scale in Sf900II serum-free medium(Invitrogen) in a 10 L bioreactor tank (27° C., 130 rpm, 50% dissolvedoxygen for 48 hours).

3. Recombinant Protein Purification

Recombinant proteins are purified from E. coli and/or insect cells usinga variety of standard chromatography methods. Briefly, cells are lysedusing sonication or detergents. The insoluble material is pelleted bycentrifugation at 10,000×g for 15 minutes. The supernatant is applied toan appropriate affinity column, e.g. His-tagged proteins are separatedusing a pre-packed chelating sepharose column (Pharmacia) or GST-taggedproteins are separated using a glutathione sepharose column (Pharmacia).After using the affinity column, proteins are further separated usingvarious techniques, such as ion exchange chromatography (columns fromPharmacia) to separate on the basis of electrical charge or sizeexclusion chromatography (columns from Tosohaas) to separate on thebasis of molecular weight, size and shape.

Expression and purification of the protein are also achieved usingeither a mammalian cell expression system or an insect cell expressionsystem. The pUB6/V5-His vector system (Invitrogen, Calif.) is used toexpress GSCC in CHO cells. The vector contains the selectable bsd gene,multiple cloning sites, the promoter/enhancer sequence from the humanubiquitin C gene, a C-terminal V5 epitope for antibody detection withanti-V5 antibodies, and a C-terminal polyhistidine (6.times.His)sequence for rapid purification on PROBOND resin (Invitrogen, Calif.).Transformed cells are selected on media containing blasticidin.

Spodoptera frugiperda (Sf9) insect cells are infected with recombinantAutographica californica nuclear polyhedrosis virus (baculovirus). Thepolyhedrin gene is replaced with the cDNA by homologous recombinationand the polyhedrin promoter drives cDNA transcription. The protein issynthesized as a fusion protein with 6×his which enables purification asdescribed above. Purified protein is used in the following activity andto make antibodies

4. Chemical Synthesis of Peptides

Proteins or portions thereof may be produced not only by recombinantmethods, but also by using chemical methods well known in the art. Solidphase peptide synthesis may be carried out in a batchwise or continuousflow process which sequentially adds .alpha.-amino- and sidechain-protected amino acid residues to an insoluble polymeric supportvia a linker group. A linker group such as methylamine-derivatizedpolyethylene glycol is attached to poly(styrene-co-divinylbenzene) toform the support resin. The amino acid residues are N-a-protected byacid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected aminoacid is coupled to the amine of the linker group to anchor the residueto the solid phase support resin. Trifluoroacetic acid or piperidine areused to remove the protecting group in the case of Boc or Fmoc,respectively. Each additional amino acid is added to the anchoredresidue using a coupling agent or pre-activated amino acid derivative,and the resin is washed. The full length peptide is synthesized bysequential deprotection, coupling of derivitized amino acids, andwashing with dichloromethane and/or N,N-dimethylformamide. The peptideis cleaved between the peptide carboxy terminus and the linker group toyield a peptide acid or amide. (Novabiochem 1997/98 Catalog and PeptideSynthesis Handbook, San Diego Calif. pp. S1-S20). Automated synthesismay also be carried out on machines such as the 431A peptide synthesizer(ABI). A protein or portion thereof may be purified by preparative highperformance liquid chromatography and its composition confirmed by aminoacid analysis or by sequencing (Creighton (1984) Proteins, Structuresand Molecular Properties, W H Freeman, New York N.Y.).

5. Antibody Development Polyclonal Antibody Preparations:

Polyclonal antibodies against recombinant proteins are raised in rabbits(Green Mountain Antibodies, Burlington, Vt.). Briefly, two New Zealandrabbits are immunized with 0.1 mg of antigen in complete Freund'sadjuvant. Subsequent immunizations are carried out using 0.05 mg ofantigen in incomplete Freund's adjuvant at days 14, 21 and 49. Bleedsare collected and screened for recognition of the antigen by solid phaseELISA and western blot analysis. The IgG fraction is separated bycentrifugation at 20,000×g for 20 minutes followed by a 50% ammoniumsulfate cut. The pelleted protein is resuspended in 5 mM Tris andseparated by ion exchange chromatography. Fractions are pooled based onIgG content. Antigen-specific antibody is affinity purified using PierceAminoLink resin coupled to the appropriate antigen.

Isolation of Antibody Fragments Directed Against CCATs from a Library ofscFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstCCAT to which the donor may or may not have been exposed (see e.g., U.S.Pat. No. 5,885,793 incorporated herein by reference in its entirety).

Rescue of the Library: A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 .mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of0.8 with shaking. Five ml of this culture is used to innoculate 50 ml of2.times.TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III,see PCT publication WO 92/01047) are added and the culture incubated at37° C. for 45 minutes without shaking and then at 37° C. for 45 minuteswith shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. andthe pellet resuspended in 2 liters of 2×TY containing 100 .mu.g/mlampicillin and 50 ug/ml kanamycin and grown overnight. Phage areprepared as described in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 .mu.g ampicillin/ml and25.mu.g kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37°C. Phagre particles are purified and concentrated from the culturemedium by two PEG-precipitations (Sambrook et al., 2001), resuspended in2 ml PBS and passed through a 0.45 .mu.m filter (Minisart NML;Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

Panning of the Library: Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phages are then used to infect 10 ml of mid-log E.coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37°C. The E. coli are then plated on TYE plates containing 1% glucose and100 .mu.g/ml ampicillin. The resulting bacterial library is then rescuedwith delta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders: Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 .mu.g/ml ofthe polypeptide of the present invention in 50 mM bicarbonate pH 9.6.Clones positive in ELISA are further characterized by PCR fingerprinting(see, e.g., PCT publication WO 92/01047) and then by sequencing.

Monoclonal Antibody Generation

i) Materials:

1) Complete Media No Sera (CMNS) for washing of the myeloma and spleencells; Hybridoma medium CM-HAT {Cell Mab (BD), 10% FBS (or HS); 5%Origen HCF (hybridoma cloning factor) containing 4 mM L-glutamine andantibiotics} to be used for plating hybridomas after the fusion.

2) Hybridoma medium CM-HT (NO AMINOPTERIN) (Cell Mab (BD), 10% FBS 5%Origen HCF containing 4 mM L-glutamine and antibiotics) to be used forfusion maintenance are stored in the refrigerator at 4-6° C. The fusionsare fed on days 4, 8, and 12, and subsequent passages. Inactivated andpre-filtered commercial Fetal Bovine serum (FBS) or Horse Serum (HS) arethawed and stored in the refrigerator at 4° C. and must be pretested formyeloma growth from single cells.

3) The L-glntamine (200 mM, 100× solution), which is stored at −20° C.freezer, is thawed and warmed until completely in solution. TheL-glntamin is dispensed into media to supplement growth. L-glntamin isadded to 2 mM for myelomas, and 4 mM for hybridoma media. Further thePenicillin, Streptomycin, Amphotericin (antibacterial-antifungal storedat −20° C.) is thawed and added to Cell Mab Media to 1%.

4) Myeloma growth media is Cell Mab Media (Cell Mab Media, Quantum Yieldfrom BD is stored in the refrigerator at 4° C. in the dark) which areadded L-glntamine to 2 mM and antibiotic/antimycotic solution to 1% andis called CMNS.

5) 1 bottle of PEG 1500 in Hepes (Roche, N.J.)

6) 8-Azaguanine is stored as the dried powder supplied by SIGMA at −700°C. until needed. Reconstitute 1 vial/500 ml of media and add entirecontents to 500 ml media (eg. 2 vials/litre).

7) Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1×) storedin the refrigerator at 4° C.

8) Clonal cell medium D (Stemcell, Vancouver) contains HAT and methylcellulose for semi-solid direct cloning from the fusion. This comes in90 ml bottles with a CoA and must be “melted at 37° C. in a waterbath inthe morning of the day of the fusion. Loosen the cap and leave in CO2incubator to sufficiently gas the medium D and bring the pH down.

9) Hybridoma supplements HT [hypoxanthine, thymidine] are to be used inmedium for the section of hybridomas and maintenance of hybridomasthrough the cloning stages respectively.

10) Origen HCF can be obtained directly from Igen and is a cellsupernatant produced from a macrophage-like cell-line. It can be thawedand aliquoted to 15 ml tubes at 5 ml per tube and stored frozen at −20°C. Positive Hybridomas are fed HCF through the first subcloning and aregradually weaned. It is not necessary to continue to supplement unlessyou have a particularly difficult hybridoma clone. This and otheradditives have been shown to be more effective in promoting newhybridoma growth than conventional feeder layers.

ii) Procedure

To generate monoclonal antibodies, mice are immunized with 5-50 ug ofantigen either intra-peritoneally (i.p.) or by intravenous injection inthe tail vein (i.v.). Typically, the antigen used is a recombinantprotein that is generated as described above. The primary immunizationtakes place 2 months prior to the harvesting of splenocytes from themouse and the immunization is typically boosted by i.v. injection of5-50 ug of antigen every two weeks. At least one week prior to expectedfusion date, a fresh vial of myeloma cells is thawed and cultured.Several flasks at different densities are maintained in order that aculture at the optimum density is ensured at the time of fusion. Theoptimum density is determined to be 3-6×10⁵ cells/ml. Two to five daysbefore the scheduled fusion, a final immunization is administered of ˜5ug of antigen in PBS i.p. or i.v.

Myeloma cells are washed with 30 ml serum free media by centrifugationat 500 g at 4° C. for 5 minutes. Viable cell density is determined inresuspended cells using hemocytometry and vital stains. Cellsresuspended in complete growth medium are stored at 37° C. during thepreparation of splenocytes. Meanwhile, to test aminopterin sensitivity,1×10⁶ myeloma cells are transferred to a 15 ml conical tube andcentrifuged at 500 g at 4° C. for 5 minutes. The resulting pellet isresuspended in 15 ml of HAT media and cells plated at 2 drops/well on a96 well plate.

To prepare splenocytes from immunized mice, the animals are euthanisedand submerged in 70% ETOH. Under sterile conditions, the spleen issurgically removed and placed in 10 ml of RPMI medium supplemented with20% fetal calf serum in a Petri dish. Cells are extricated from thespleen by infusing the organ with medium >50 times using a 21 g syringe.

Cells are harvested and washed by centrifugation (at 500 g at 4° C. for5 minutes) with 30 ml of medium. Cells are resuspended in 10 ml ofmedium and the density of viable cells determined by hemocytometry usingvital stains. The splenocytes are mixed with myeloma cells at a ratio of5:1 (spleen cells: myeloma cells). Both the myeloma and spleen cells arewashed 2 more times with 30 ml of RPMI-CMNS. Spin at 800 rpm for 12minutes.

Supernatant is removed and cells are resuspended in 5 ml of RPMI-CMNSand are pooled to fill volume to 30 ml and spin down as before. Then,the pellet is broken up by gently tapping on the flow hood surface andresuspended in 1 ml of BMB REG1500 (prewarmed to 37° C.) dropwise with 1cc needle over 1 minute.

RPMI-CMNS to the PEG cells and RPMI-CMNS are added to slowly dilute outthe PEG. Cells are centrifuged and diluted in 5 ml of Complete media and95 ml of Clonacell Medium D (HAT) media (with 5 ml of HCF). The cellsare plated out 10 ml per small petri plate.

Myeloma/HAT control. P is prepared as follows: dilute about 1000 P3X63Ag8.653 myeloma cells into 1 ml of mediu D and transfer into a singlewell of a 24 well plate. Plates are placed in incubator, with two platesinside of a large petri plate, with an additional petri plate full ofdistilled water, for 10-18 days under 5% CO2 overlay at 37° C. Clonesare picked from semisolid agarose into 96 well plates containing 150-200ul of CM-HT. Supernatants are screened 4 days later in ELISA, andpositive clones are moved up to 24 well plates. Heavy growth willrequire changing of the media at day 8 (+/−150 ml). One should furtherdecrease the HCF to 0.5% (gradually—2%, then 1%, then 0.5%) in thecloning plates.

For further references see Kohler G, and C. Milstein Continuous culturesof fused cells secreting antibody of predefined specificity. 1975.Nature 256: 495-497; Lane, R. D. A short duration polyethylene glycolfusion technique for increasing production of monoclonalantibody-secreting hybridomas. 1985. J. Immunol. Meth. 81:223-228;

Harlow, E. and D. Lane. Antibodies: A laboratory manual. Cold SpringHarbour Laboratory Press. 1988; Kubitz, D. The Scripps ResearchInstitute. La Jolla. Personal Communication; Zhong, G., Berry, J. D.,and Choukri, S. (1996) Mapping epitopes of Chlamydia trachomatisneutralizing monoclonal antibodies using phage random peptide libraries.J. Indust. Microbiol. Biotech. 19, 71-76; Berry, J. D., Licea, A.,Popkov, M., Cortez, X., Fuller, R., Elia, M., Kerwin, L., and C. F.Barbas III. (2003) Rapid monoclonal antibody generation via dendriticcell targeting in vivo. Hybridoma and Hybridomics 22 (1), 23-31.

6. mRNA Expression

Validation in Tissues by Taqman

Expression of mRNA is quantitated by RT-PCR using TaqMan® technology.The Taqman system couples a 5′ fluorogenic nuclease assay with PCR forreal time quantitation. A probe is used to monitor the formation of theamplification product.

Total RNA is isolated from cancer model cell lines using the RNEasy Kit®(Qiagen) per manufacturer's instructions and included DNase treatment.Normal human tissue RNAs are acquired from commercial vendors (Ambion,Austin, Tex.; Stratagene, La Jolla, Calif., BioChain Institute,Newington, N.H.) as were RNAs from matched disease/normal tissues.

Target transcript sequences are identified for the differentiallyexpressed peptides by searching the BlastP database. TaqMan assays (PCRprimer/probe set) specific for those transcripts are identified bysearching the Celera Discovery System™ (CDS) database. The assays aredesigned to span exon-exon borders and do not amplify genomic DNA.

The TaqMan primers and probe sequences are as designed by AppliedBiosystems (AB) as part of the Assays on Demand™ product line or bycustom design through the AB Assays by Design℠ service.

RT-PCR is accomplished using AmpliTaqGold and MultiScribe reversetranscriptase in the One Step RT-PCR Master Mix reagent kit (AB)according to the manufacturer's instructions. Probe and primerconcentrations are 250 nM and 900 nM, respectively, in a 15 Ξl reaction.For each experiment, a master mix of the above components is made andaliquoted into each optical reaction well. Eight nanograms of total RNAis the template. Each sample is assayed in triplicate. QuantitativeRT-PCR is performed using the ABI Prism® 7900HT Sequence DetectionSystem (SDS). Cycling parameters follow: 48° C. for 30 min. for onecycle; 95° C. for 10 min for one cycle; 95° C. for 15 sec, 60° C. for 1min. for 40 cycles.

The SDS software calculates the threshold cycle (C_(T)) for eachreaction, and C_(T) values are used to quantitate the relative amount ofstarting template in the reaction. The C_(T) values for each set ofthree reactions are averaged for all subsequent calculations

Data are analyzed for fold difference in expression using an endogenouscontrol for normalization and is expressed relative to a normal tissueor normal cell line reference. The choice of endogenous control isdetermined empirically by testing various candidates against the cellline and tissue RNA panels and selecting the one with the leastvariation in expression. Relative changes in expression are quantitatedusing the 2^(−ΔΔCT) Method. Livak, K. J. and Schmittgen, T. D. (2001)Methods 25: 402-408; User bulletin #2: ABI Prism 7700 Sequence DetectionSystem.

Validation by Tissue Flow Cytometry Analysis

Post tissue processing, cells are sorted by flow cytometry known in theart to enrich for epithelial cells. Alternatively, cells isolated fromlung tissue are stained directly with EpCAM (for epithelial cells) andthe specific antibody to CCAT. Cell numbers and viability are determinedby PI exclusion (GUAVA) for cells isolated from both normal and tumorlung tissue. A minimum of 0.5×10⁶ cells are used for each analysis.Cells are washed once with Flow Staining Buffer (0.5% BSA, 0.05% NaN3 inD-PBS). To the cells, 20 ul of each antibody for CCAT are added. Anadditional 5 ul of EpCAM antibody conjugated to APC were added whenunsorted cells are used in the experiment. Cells are incubated withantibodies for 30 minutes at 4° C. Cells are washed once with FlowStaining Buffer and either analyzed immediately on the LSR flowcytometry apparatus or fixed in 1% formaldehyde and store at 4° C. untilLSR analysis. The antibodies used to detect CCAT targets are allpurchased by BD Biosciences and PE-conjugated. The isotype controlantibody used for these experiments is PE-conjugated mouse IgG1k.

7. Detection and Diagnosis of CCAT by Liquid Chromatography and MassSpectrometry (LC/MS)

The proteins from cells can be prepared by reduction, alkylation andcysteine-containing peptide enrichment of concentrated conditionedmedia.

The differential expression of proteins in disease and healthy samplesare quantitated using Liquid Chromatography Mass Spectrometry. The LC/MSspectra from disease and healthy (control) samples are collected andprocessed using the following steps:

The raw scans from the LC/MS instrument are subjected to peak detectionand noise reduction software. Filtered peak lists are then used todetect ‘features’ corresponding to specific peptides from the originalsample(s). Features are characterized by their mass/charge, charge,retention time, isotope pattern and intensity.

Similar experiments are repeated in order to increase the confidence indetection of a peptide. These multiple acquisitions are computationallyaggregated into one experiment. The intensity of a peptide present inboth healthy and disease samples is used to calculate the differentialexpression, or relative abundance, of the peptide. The intensity of apeptide found exclusively in one sample is used to calculate atheoretical expression ratio for that peptide (singleton). Expressionratios are calculated for each peptide of each replicate of theexperiment

Statistical tests are performed to assess the robustness of the data andselect statistically significant differentials. To assess generalquality of the data, a) ensured that similar features are detected inall replicates of the experiment; b) number of matched ions betweenreplicates; c) calculated the overall pair wise intensity correlationsbetween LC/MS maps of process replicates to ensure that the expressionratios for peptides are reproducible across the multiple replicates; d)aggregated multiple experiments in order to compare the expression ratioof a peptide in multiple diseases or disease samples.

8. Expression Validation by IHC in Tissue Sections Tissue Sections

Paraffin embedded, fixed tissue sections are obtained from a panel ofnormal tissues (Adrenal, Bladder, Lymphocytes, Bone Marrow, Breast,Cerebellum, Cerebral cortex, Colon, Endothelium, Eye, Fallopian tube,Small Intestine, Heart, Kidney [glomerulus, tubule], Liver, Lung, Testesand Thyroid) as well as 30 tumor samples with matched normal adjacenttissues from pancreas, lung, colon, prostate, ovarian and breast. Inaddition, other tissues are selected for testing such as bladder renal,hepatocellular, pharyngeal and gastric tumor tissues. Replicate sectionsare also obtained from numerous tumor types (Bladder Cancer, LungCancer, Breast Cancer, Melanoma, Colon Cancer, Non-Hodgkins Lymphoma,Endometrial Cancer, Ovarian Cancer, Head and Neck Cancer, ProstateCancer, Leukemia [ALL and CML] and Rectal Cancer). Sections are stainedwith hemotoxylin and eosin and histologically examined to ensureadequate representation of cell types in each tissue section.

An identical set of tissues will be obtained from frozen sections andare used in those instances where it is not possible to generateantibodies that are suitable for fixed sections. Frozen tissues do notrequire an antigen retrieval step.

Paraffin Fixed Tissue Sections

Hemotoxylin and Eosin staining of paraffin embedded, fixed tissuesections.

Sections are deparaffinized in 3 changes of xylene or xylene substitutefor 2-5 minutes each. Sections are rinsed in 2 changes of absolutealcohol for 1-2 minutes each, in 95% alcohol for 1 minute, followed by80% alcohol for 1 minute. Slides are washed well in running water andstained in Gill solution 3 hemotoxylin for 3 to 5 minutes. Following avigorous wash in running water for 1 minute, sections are stained inScott's solution for 2 minutes. Sections are washed for 1 min in runningwater then conterstained in Eosin solution for 2-3 minutes dependingupon development of desired staining intensity. Following a brief washin 95% alcohol, sections are dehydrated in three changes of absolutealcohol for 1 minute each and three changes of xylene or xylenesubstitute for 1-2 minutes each. Slides are coverslipped and stored foranalysis.

Optimisation of Antibody Staining

or each antibody, a positive and negative control sample are generatedusing data from the ICAT analysis of the colon cancer celllines/tissues. Cells are selected that are known to express low levelsof a particular target as determined from the ICAT data. This cell lineis the reference normal control. Similarly, a colon tumor line isselected that is determined to overexpress the target is selected.

Antigen Retrieval

Sections are deparaffinized and rehydrated by washing 3 times for 5minutes in xylene; two times for 5 minutes in 100% ethanol; two timesfor 5 minutes in 95% ethanol; and once for 5 minutes in 80% ethanol.Sections are then placed in endogenous blocking solution (methanol+2%hydrogen peroxide) and incubated for 20 minutes at room temperature.Sections are rinsed twice for 5 minutes each in deionized water andtwice for 5 minutes in phosphate buffered saline (PBS), pH 7.4.Alternatively, where necessary sections are deparrafinized by HighEnergy Antigen Retrieval as follows: sections are washed three times for5 minutes in xylene; two times for 5 minutes in 100% ethanol; two timesfor 5 minutes in 95% ethanol; and once for 5 minutes in 80% ethanol.Sections are placed in a Coplin jar with dilute antigen retrievalsolution (10 mM citrate acid, pH 6). The Coplin jar containing slides isplaced in a vessel filled with water and microwaved on high for 2-3minutes (700 watt oven). Following cooling for 2-3 minutes, steps 3 and4 are repeated four times (depending on tissue), followed by cooling for20 minutes at room temperature. Sections are then rinsed in deionizedwater, two times for 5 minutes, placed in modified endogenous oxidationblocking solution (PBS+2% hydrogen peroxide).and rinsed for 5 minutes inPBS.

Blocking and Staining

Sections are blocked with PBS/1% bovine serum albumin (PBA) for 1 hourat room temperature followed by incubation in normal serum diluted inPBA (2%) for 30 minutes at room temperature to reduce non-specificbinding of antibody. Incubations are performed in a sealed humiditychamber to prevent air-drying of the tissue sections. (The choice ofblocking serum is the same as the species of the biotinylated secondaryantibody). Excess antibody is gently removed by shaking and sectionscovered with primary antibody diluted in PBA and incubated either atroom temperature for 1 hour or overnight at 4° C. (Care is taken thatthe sections do not touch during incubation). Sections are rinsed twicefor 5 minutes in PBS, shaking gently. Excess PBS is removed by gentlyshaking. The sections are covered with diluted biotinylated secondaryantibody in PBA and incubated for 30 minutes to 1 hour at roomtemperature in the humidity chamber. If using a monoclonal primaryantibody, addition of 2% rat serum is used to decrease the background onrat tissue sections. Following incubation, sections are rinsed twice for5 minutes in PBS, shaking gently. Excess PBS is removed and sectionsincubated for 1 hour at room temperature in Vectastain ABC reagent (asper kit instructions). The lid of the humidity chamber is secured duringall incunations to ensure a moist environment. Sections are rinsed twicefor 5 minutes in PBS, shaking gently.

Develop and Counterstain

Sections are incubated for 2 minutes in peroxidase substrate solutionthat is made up immediately prior to use as follows:

-   -   10 mg diaminobenzidine (DAB) dissolved in 10 ml 50 mM sodium        phosphate buffer, pH 7.4.    -   12.5 microliters 3% CoCl2/NiCl2 in deionized water    -   1.25 microliters hydrogen peroxide

Slides are rinsed well three times for 10 min in deionized water andcounterstained with 0.01% Light Green acidified with 0.01% acetic acidfor 1-2 minutes depending on intensity of counterstain desired.

Slides are rinsed three times for 5 minutes with deionized water anddehydrated two times for 2 minutes in 95% ethanol; two times for 2minutes in 100% ethanol; and two times for 2 minutes in xylene. Stainedslides are mounted for visualization by microscopy.

9. IHC Staining of Frozen Tissue Sections

Fresh tissues are embedded carefully in OCT in plastic mold, withouttrapping air bubbles surrounding the tissue. Tissues are frozen bysetting the mold on top of liquid nitrogen until 70-80% of the blockturns white at which point the mold is placed on dry ice. The frozenblocks were stored at −80° C. Blocks are sectioned with a cryostat withcare taken to avoid warming to greater than −10° C. Initially, the blockis equilibrated in the cryostat for about 5 minutes and 6-10 mm sectionsare cut sequentially. Sections are allowed to dry for at least 30minutes at room temperature. Following drying, tissues are stored at 4°C. for short term and −80° C. for long term storage.)

Sections are fixed by immersing in acetone jar for 1-2 minutes at roomtemperature, followed by drying at room temp. Primary antibody is added(diluted in 0.05 M Tris-saline [0.05 M Tris, 0.15 M NaCl, pH 7.4], 2.5%serum) directly to the sections by covering the section dropwise tocover the tissue entirely. Binding is carried out by incubation achamber for 1 hour at room temperature. Without letting the sections dryout, the secondary antibody (diluted in Tris-saline/2.5% serum) is addedin a similar manner to the primary and incubated as before (at least 45minutes).

Following incubation, the sections are washed gently in Tris-saline for3-5 minutes and then in Tris-saline/2.5% serum for another 3-5 minutes.If a biotinylated primary antibody is used, in place of the secondaryantibody incubation, slides are covered with 100 ul of diluted alkalinephosphatase conjugated streptavidin, incubated for 30 minutes at roomtemperature and washed as above. Sections are incubated with alkalinephosphatse substrate (1 mg/ml Fast Violet; 0.2 mg/ml Napthol AS-MXphosphate in Tris-Saline pH 8.5) for 10-20 minutes until the desiredpositive staining is achieved at which point the reaction is stopped bywashing twice with Tris-saline. Slides are counter-stained with Mayer'shematoxylin for 30 seconds and washed with tap water for 2-5 minutes.Sections are mounted with Mount coverslips and mounting media.

10. Assay for Antibody Dependent Cellular Cytotoxicity

Cultured tumor cells are labeled with 100 μCi 51Cr for 1 hour;Livingston, P. O., Zhang, S., Adluri, S., Yao, T.-J., Graeber, L.,Ragupathi, G., Helling, F., & Fleischer, M. (1997). Cancer Immunol.Immunother. 43, 324-330. After being washed three times with culturemedium, cells are resuspended at 10⁵/ml, and 100 μl/well are plated onto96-well round-bottom plates. A range of antibody concentrations areapplied to the wells, including an isotype control together with donorperipheral blood mononuclear cells that are plated at a 100:1 and 50:1ratio. After an 18-h incubation at 37° C., supernatant (30 μl/well) isharvested and transferred onto Lumaplate 96 (Packard), dried, and readin a Packard Top-Count NXT γ counter. Each measurement is carried out intriplicate. Spontaneous release is determined by cpm of tumor cellsincubated with medium and maximum release by cpm of tumor cells plus 1%Triton X-100 (Sigma). Specific lysis is defined as: % specificlysis=[(experimental release−spontaneous release)/(maximumrelease−spontaneous release)]×100. The percent ADCC is expressed as peakspecific lysis postimmune subtracted by preimmune percent specificlysis. A doubling of the ADCC to >20% is considered significant.

11. Assay for Complement Dependent Cytotoxicity

Chromium release assays to assess complement-mediated cytotoxicity areperformed for each patient at various time points; Dickler, M. N.,Ragupathi, G., Liu, N. X., Musselli, C., Martino, D. J., Miller, V. A.,Kris, M. G., Brezicka, F. T., Livingston, P. O. & Grant, S. C. (1999)Clin. Cancer Res. 5, 2773-2779. Cultured tumor cells are washed inFCS-free media two times, resuspended in 500 μl of media, and incubatedwith 100 μCi ⁵¹Cr per 10 million cells for 2 h at 37° C. The cells arethen shaken every 15 min for 2 h, washed 3 times in media to achieve aconcentration of approximately 20,000 cells/well, and then plated inround-bottom plates. The plates contain either 50 μl cells plus 50 μlmonoclonal antibody, 50 μl cells plus serum (pre- and posttherapy), or50 μl cells plus mouse serum as a control. The plates are incubated in acold room on a shaker for 45 min. Human complement of a 1:5 dilution(resuspended in 1 ml of ice-cold water and diluted with 3% human serumalbumin) is added to each well at a volume of 100 Control wells includethose for maximum release of isotope in 10% Triton X-100 (Sigma) and forspontaneous release in the absence of complement with medium alone. Theplates are incubated for 2 h at 37° C., centrifuged for 3 min, and then100 μl of supernatant is removed for radioactivity counting. Thepercentage of specific lysis is calculated as follows: %cytotoxicity=[(experimental release-spontaneous release)/(maximumrelease-spontaneous release)]×100. A doubling of the CDC to >20% isconsidered significant.

12. In Vitro Assays in Cell Lines; RNAi

Lipofectamine is purchased from Invitrogen (Carlsbad, Calif.) andGeneSilencer from Gene Therapy Systems (San Diego, Calif.). SyntheticsiRNA oligonucleotides are from Dharmacon (Lafayette, Colo.), Qiagen(Valencia, Calif.) or Ambion (Austin. Tex.) RNeasy 96 Kit is purchasedfrom Qiagen (Valencia, Calif.). Apopone homogeneous caspase-3/7 kit andCellTiter 96 AQueous One Solution Cell Proliferation Assay are bothpurchased from Promega (Madison, Wis.). Alamar Blue proliferation assaycan be purchased from Biosource (Camarillo, Calif.). Function blockingantibodies are purchased from Chemicon (Temecula, Calif.), Biotrend(Cologne, Germany) or Alexis Corporation (San Diego, Calif.). Cellinvasion assay kits from purchased from Chemicon (Temecula, Calif.).RiboGreen RNA Quantitation Kit is purchased from Molecular probes(Eugene, Oreg.).

RNAi

RNAi is performed by using Smartpools (Dharmacon), 4—for Silencing siRNAduplexes (Qiagen) or scrambled negative control siRNA (Ambion).Transient transfections are carried out in triplicate by using eitherLipofectamine 2000 from Invitrogen (Carlsbad, Calif.) or by usingGeneSilencer from Gene Therapy Systems (San Diego, Calif.) in methodsdescribed below. 1 to 4 days after transfections, total RNA is isolatedby using the RNeasy 96 Kit (Qiagen) according to manufacturer'sinstructions and expression of mRNA is quantitated by using TaqMantechnology. Protein expression levels are examined by flow cytometry andapoptosis and proliferation assays are performed daily using Apop-onehomogeneous caspase-3/7 kit and CellTiter 96 AQueous One Solution CellProliferation Assay (see protocols below).

i) RNAi Transfections—Lipofectamine 2000

Transient transfections are carried out on sub-confluent colon cancercell lines as previously described. Elbashir, S. M. et al. (2001) Nature411: 494-498; Caplen, N.J. et al. (2001) Proc Natl Acad Sci USA 98:9742-9747; Sharp, P. A. (2001) Genes and Development 15: 485-490.Synthetic RNA to gene of interest or scrambled negative control siRNA istransfected using lipofectamine according to manufacturer'sinstructions. Cells are plated in 96 well plates in antibiotics freemedium. The next day, the transfection reagent and siRNA are preparedfor transfections as follows: Each 0.1-1 ul of lipofectamine 2000 and10-150 mM siRNA are resuspended 25 ul serum-free media and incubated atroom temperature for 5 minutes. After incubation, the diluted siRNA andthe lipofectamine 2000 are combined and incubated for 20 minutes at roomtemperature. The cells are then washed and the combinedsiRNA-Lipofectamine 2000 reagent added. After further 4 hoursincubation, 50 ul serum containing medium is added to each well. 1 and 4days after transfection, expression of mRNA is quantitated by RT-PCRusing TaqMan technology and protein expression levels are examined byflow cytometry. Apoptosis and proliferation assays are performed dailyusing Apop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous OneSolution Cell Proliferation Assay (see protocols below).

ii) RNAi Transfections—GeneSilencer

Transient transfections are carried out on sub-confluent colon cancercell lines as previously described. Elbashir, S. M. et al. (2001) Nature411: 494-498; Caplen, N.J. et al. (2001) Proc Natl Acad Sci USA 98:9742-9747; Sharp, P. A. (2001) Genes and Development 15: 485-490.Synthetic RNA to gene of interest or scrambled negative control siRNA istransfected using GeneSilencer according to manufacturer's instructions.Cells are plated in 96 well plates in antibiotics free medium. The nextday, the transfection reagent and the synthetic siRNA are prepared fortransfections as follows: predetermined amount of Gene Silencer isdiluted in serum-free media to a final volume of 20 ul per well. Afterresuspending 10-150 mM siRNA in 20 ul serum-free media, the reagents arecombined and incubated at room temperature for 5-20 minutes. Afterincubation, the siRNA-Gene Silencer reagent is added to each well andincubated in a 37° C. incubator for 4 hours before an equal volume ofserum containing media is added back to the cultured cells. The cellsare then incubated for 1 to 4 days before mRNA, protein expression andeffects on apoptosis and proliferation are examined.

Testing of Function Blocking Antibodies

Sub-confluent colon cancer cell lines are serum-staved overnight. Thenext day, serum-containing media is added back to the cells in thepresence of 5-50 ng/ml of function blocking antibodies. After 2 or 5days incubation at 37° C. 5% CO2, antibody binding is examined by flowcytometry and apoptosis and proliferation are examined by usingprotocols described below.

Apoptosis

Apoptosis assay is performed by using the Apop-one homogeneouscaspase-3/7 kit from Promega. Briefly, the caspase-3/7 substrate isthawed to room temperature and diluted 1:100 with buffer. The dilutedsubstrate is then added 1:1 to cells, control or blank. The plates arethen placed on a plate shaker for 30 minutes to 18 hours at 300-500 rpm.The fluorescence of each well is then measured at using an excitationwavelength of 485+/−20 nm and an emission wavelength of 530+/−25 nm.

Proliferation—MTS

Proliferation assay is performed by using the CellTiter 96 AQueous OneSolution Cell Proliferation Assay kit from Promega. 20 ul of CellTiter96 AQueous One Solution is added to 100 ul of culture medium. The platesare then incubated for 1-4 hours at 37° C. in a humidified 5% CO2incubator. After incubation, the change in absorbance is read at 490 nm.

Proliferation—Alamar Blue

Proliferation assay is performed by using the Alamar Blue assay fromBiosource. 10 ul of Alamar Blue reagent is added to 100 ul of cells inculture medium. The plates are then incubated for 1-4 hours at 37° C. ina humidified 5% CO2 incubator. After incubation, the change influorescence is measured at using an excitation wavelength of 530 nm andan emission wavelength of 595 nm.

Cell Invasion

Cell invasion assay is performed by using the 96 well cell invasionassay kit available from Chemicon. After the cell invasion chamberplates are adjusted to room temperature, 100 ul serum-free media isadded to the interior of the inserts. 1-2 hours later, cell suspensionsof 1×10⁶ cells/ml are prepared. Media is then carefully removed from theinserts and 100 ul of prepared cells are added into the insert +/−0 to50 ng function blocking antibodies. The cells are pre-incubated for 15minutes at 37° C. before 150 ul of media containing 10% FBS is added tothe lower chamber. The cells are then incubated for 48 hours at 37° C.After incubation, the cells from the top side of the insert arediscarded and the invasion chamber plates are then placed on a new96-well feeder tray containing 150 ul of pre-warmed cell detachmentsolution in the wells. The plates are incubated for 30 minutes at 37° C.and are periodically shaken. Lysis buffer/dye solution (4 ul CyQuantDye/300 ul 4× lysis buffer) is prepared and added to each well ofdissociation buffer/cells on feeder tray. The plates are incubated for15 minutes at room temperature before 150 ul is transferred to a new96-well plate. Fluorescence of invading cells is then read at 480excitation and 520 emission.

Receptor Internalization

For quantification of receptor internalization, ELISA assays areperformed essentially as described by Daunt et al. Daunt, D. A., Hurtz,C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol.Pharmacol. 51, 711-720. The cell lines are plated at 6×10⁵ cells per ina 24-well tissue culture dishes that have previously been coated with0.1 mg/ml poly-L-lysine. The next day, the cells are washed once withPBS and incubated in DMEM at 37° C. for several minutes. Agonist to thecell surface target of interest is then added at a pre-determinedconcentration in prewarmed DMEM to the wells. The cells are thenincubated for various times at 37° C. and reactions are stopped byremoving the media and fixing the cells in 3.7% formaldehyde/TBS for 5min at room temperature. The cells are then washed three times with TBSand nonspecific binding blocked with TBS containing 1% BSA for 45 min atroom temperature. The first antibody is added at a pre-determineddilution in TBS/BSA for 1 h at room temperature. Three washes with TBSfollowed, and cells are briefly reblocked for 15 min at roomtemperature. Incubation with goat anti-mouse conjugated alkalinephosphatase (Bio-Rad) diluted 1:1000 in TBS/BSA is carried out for 1 hat room temperature. The cells are washed three times with TB S and acolorimetric alkaline phosphatase substrate is added. When the adequatecolor change is reached, 100-0 samples are taken for colorimetricreadings.

mRNA Expression

Expression of mRNA is quantitated by RT-PCR using TaqMan® technology.Total RNA is isolated from cancer model cell lines using the RNEasy 96kit (Qiagen) per manufacturer's instructions and included DNasetreatment. Target transcript sequences are identified for thedifferentially expressed peptides by searching the BlastP database.TaqMan assays (PCR primer/probe set) specific for those transcripts areidentified by searching the Celera Discovery System™ (CDS) database. Theassays are designed to span exon-exon borders and do not amplify genomicDNA. The TaqMan primers and probe sequences are as designed by AppliedBiosystems (AB) as part of the Assays on Demand™ product line or bycustom design through the AB Assays by Design℠ service. RT-PCR isaccomplished using AmpliTaqGold and MultiScribe reverse transcriptase inthe One Step RT-PCR Master Mix reagent kit (AB) according to themanufacturer's instructions. Probe and primer concentrations are 900 nMand 250 nM, respectively, in a 250 reaction. For each experiment, amaster mix of the above components is made and aliquoted into eachoptical reaction well. 5 ul of total RNA is the template. Each sample isassayed in triplicate. Quantitative RT-PCR is performed using the ABIPrism® 7900HT Sequence Detection System (SDS). Cycling parametersfollow: 48° C. for 30 min. for one cycle; 95° C. for 10 min for onecycle; 95° C. for 15 sec, 60° C. for 1 min. for 40 cycles.

The SDS software calculates the threshold cycle (C_(T)) for eachreaction, and C_(T) values are used to quantitate the relative amount ofstarting template in the reaction. The C_(T) values for each set ofthree reactions are averaged for all subsequent calculations.

Total RNA is quantitated by using RiboGreen RNA Quantitation Kitaccording to manufacturer's instructions and the % mRNA expression iscalculated using total RNA for normalization. % knockdown is thencalculated relative to the no addition control.

13. In Vivo Studies by Using Antibodies

Treatment of Colon Cancer Cells with Monoclonal Antibodies.

colon cancer cells are seeded at a density of 4×10⁴ cells per well in96-well microtiter plates and allowed to adhere for 2 hours. The cellsare then treated with different concentrations of anti-CCAT monoclonalantibody (Mab) or irrelevant isotype matched (anti-rHuIFN-. gamma. Mab)at 0.05, 0.5 or 5.0 mug/ml. After a 72 hour incubation, the cellmonolayers are stained with crystal violet dye for determination ofrelative percent viability (RPV) compared to control (untreated) cells.Each treatment group consists of replicates. Cell growth inhibition ismonitored.

Treatment of NIH 3T3 Cells Overexpression CCAT Protein with MonoclonalAntibodies.

NIH 3T3 expressing CCAT protein are treated with differentconcentrations of anti-CCAT MAbs. Cell growth inhibition is monitored.

In Vivo Treatment of NIH 3T3 Cells Overexpressing CCAT with Anti-CCATMonoclonal Antibodies.

NIH 3T3 cells transfected with either a CCAT expression plasmidor theneo-DHFR vector are injected into nu/nu (athymic) mice subcutaneously ata dose of 10⁶ cells in 0.1 ml of phosphate-buffered saline. On days 0,1, 5 and every 4 days thereafter, 100 mug (0.1 ml in PBS) of either anirrelevant or anti-CCAT monoclonal antibody of the IG2A subclass isinjected intraperitoneally. Tumor occurrence and size are monitored for1 month period of treatment.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention, which are obvious to those skilled inthe field of molecular biology or related fields, are intended to bewithin the scope of the following claims.

That which is claimed is:
 1. An isolated protein comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS:1-1233and 2228-2638.
 2. A composition comprising the protein of claim 1 and apharmaceutically acceptable carrier.
 3. An isolated nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of: a) SEQ ID NOS:1234-2227; b) nucleotide sequences thatencode a protein comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOS:1-1233 and 2228-2638; and c) nucleotidesequences that are completely complementary to the nucleotide sequencesof a) or b).
 4. An isolated RNAi or antisense nucleic acid molecule thatselectively binds to the nucleic acid molecule of claim
 3. 5. Anisolated antibody that selectively binds to the protein of claim
 1. 6.The antibody of claim 5, wherein the antibody is at least one of amonoclonal, polyclonal, fully human, humanized, chimeric, single-chain,or anti-idiotypic antibody.
 7. A cell line, hybridoma, phage, ortransgenic organism that produces the antibody of claim
 5. 8. Theantibody of claim 5, wherein the antibody is coupled to a compositionselected from the group consisting of detectable substances andtherapeutic agents.
 9. A composition comprising the antibody of claim 5and a pharmaceutically acceptable carrier.
 10. An isolated antibodyfragment of the antibody of claim 5, wherein the antibody fragmentcomprises a fragment selected from the group consisting of: a) an Fabfragment; b) an F(ab′)₂ fragment; and c) an Fv fragment.
 11. A method ofmodulating cell proliferation or apoptosis, the method comprisingcontacting a cell with the antibody of claim
 5. 12. The method of claim11, wherein the method comprises either inhibiting proliferation ofcolon cancer cells or stimulating apoptosis of colon cancer cells.
 13. Amethod of modulating cell proliferation or apoptosis, the methodcomprising contacting a cell with the RNAi or antisense nucleic acidmolecule of claim
 4. 14. A method of detecting the protein of claim 1 ina sample, the method comprising contacting the sample with an isolatedantibody that selectively binds to the protein and determining whetherthe antibody binds to the protein.
 15. A method of detecting the nucleicacid molecule of claim 3 in a sample, the method comprising contactingthe sample with an oligonucleotide that specifically hybridizes to thenucleic acid molecule and determining whether the oligonucleotide bindsto the nucleic acid molecule.
 16. A method of diagnosing, prognosing, ordetermining risk of colon cancer in a subject, the method comprisingdetecting at least one molecule in a sample, wherein the presence orabundance of the molecule is indicative of colon cancer, and wherein themolecule is selected from the group consisting of: a) proteinscomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS:1-1233 and 2228-2638; b) antibodies that selectively bind tothe protein of a); c) nucleic acid molecules comprising a nucleotidesequence selected from the group consisting of SEQ ID NOS:1234-2227 andnucleotide sequences that encode the protein of a); and d) nucleic acidmolecules comprising a nucleotide sequence that is completelycomplementary to the nucleic acid molecule of c).
 17. A method oftreating colon cancer, the method comprising administering atherapeutically effective amount of the antibody of claim 5 to asubject.
 18. A method of screening agents, the method comprisingcontacting the protein of claim 1 or a cell that expresses the proteinwith an agent, and assaying for whether the agent binds to the proteinor modulates the function, activity, or expression of the protein.
 19. Acomposition comprising the agent identified by the method of claim 18and a pharmaceutically acceptable carrier.
 20. A method of determiningor predicting the effectiveness of a treatment or selecting a treatmentfor administration to a subject having colon cancer, the methodcomprising detecting the presence, abundance, or activity of the proteinof claim 1 in a sample and determining or predicting the effectivenessof the treatment or selecting the treatment for administration based onthe presence, abundance, or activity of the protein.